Products containing polyphenol(s) and l-arginine

ABSTRACT

Foods and pharmaceuticals which contain cocoa and/or nut procyanidin(s) in combination with L-arginine are effective to induce a physiological increase in nitric oxide production in a mammal having ingested the product. A preferred food product is a confection, particularly a dark or milk chocolate containing nuts. The procyanidins may be natural or synthetic and may be provided by food ingredients such as chocolate liquor and/or cocoa solids prepared from underfermented beans and nut skins. The L-arginine may be natural or synthetic and may be provided by food ingredients such as nut meats, nut pastes, and/or nut flours, seeds, seed pastes, and/or seed flours, or gelatin. The beneficial health effects may include, for example, reduced blood pressure, resistance to cardiovascular disease, and anticancer activity.

This application is a divisional application of U.S. application Ser.No. 10/176,126 filed Jun. 19, 2002, which is a continuation applicationof U.S. application Ser. No. 09/284,783, filed Mar. 12, 1999, abandoned,which is a National Stage of International Appl. No. PCT/US99/05545,filed Mar. 12, 1999, which claims priority from U.S. application Ser.No. 09/041,327 filed Mar. 12, 1998, now abandoned; the disclosure ofeach hereby being incorporated herein by reference.

FIELD OF THE INVENTION

The invention relates to products containing polyphenols and L-argininethat have a beneficial effect on the health of mammals.

BACKGROUND OF THE INVENTION

Polyphenolic compounds are bioactive substances that are derived fromplant materials and are closely associated with the sensory andnutritional quality of products containing them.

Proanthocyanidins are a class of polyphenolic compounds found in severalplant species. They are oligomers of flavan-3-ol monomer units mostfrequently linked either as 4→6 or 4→8. The most common classes are theprocyanidins which are chains of catechin, epicatechin, and their gallicacid esters and the prodelphinidins which consist of gallocatechin,epigallocatechin, and their gallic acid esters as the monomeric units.Structural variations in proanthocyanidin oligomers may also occur withthe formation of a second interflavanoid bond by C—O oxidative couplingto form A-type oligomers. Due to the complexity of this conversion,A-type proanthocyanidins are not as frequently encountered in nature incomparison to the B-type oligomers.

The term “cocoa polyphenols” includes polyphenolic products includingproanthocyanidins, more particularly procyanidins, extracted from cocoabeans and derivatives thereof. More specifically, the term “cocoapolyphenol” includes monomers of the formula A_(n) (where n is 1) oroligomers of the formula A_(n) (where n is an integer from 2 to 18, andhigher), wherein A has the formula:

and R is 3-(α)-OH, 3-(β), 3-(α)-O-saccharide, 3-(β)-O-saccharide,3-(α)-O—C(O)—R′, or 3-(β)-O—C(O)—R′;

bonding between adjacent monomers takes place at positions 4, 6 or 8;

a bond to a monomer in position 4 has alpha or beta stereochemistry;

X, Y and Z are selected from the group consisting of A, hydrogen, and asaccharide moiety, with the proviso that as to at least one terminalmonomer, bonding of the adjacent monomer thereto is at position 4 andoptionally Y=Z=hydrogen; and

wherein the saccharide moiety is a mono- or di-saccharide moiety and maybe optionally substituted with a phenolic moiety and R′ may be an arylor heteroaryl moiety optionally substituted with at least one hydroxylgroup; and

salts, derivatives and oxidation products thereof.

Preferably, the saccharide moiety is derived from the group consistingof glucose, galactose, xylose, rhamnose and arabinose. The saccharidemoiety and any or all of R, X, Y, and Z may optionally be substituted atany position with a phenolic moiety via an ester bond. The phenolicmoiety is selected from the group consisting of caffein, cinnamic,coumaric, ferulic, gallic, hydroxybenzoic and sinapic acids.

Proanthocyanidins have attracted increasing attention due to the rapidlygrowing body of evidence associating these compounds with a wide rangeof potential health benefits. Tea catechins have recently beenassociated with potent antioxidant activity and with the reduction oftumor multiplicity in laboratory mice (Lunder, 1992; Wang et al., 1992;Chung et al., 1992). Additionally, the proanthocyanidins in grape seedextracts have been shown to have free radical scavenging abilities andto decrease the susceptibility of healthy cells to toxic andcarcinogenic agents (Bagchi et al., 1997; Waterhouse and Walzem, 1997;Joshi et al., 1998). Polyphenols in grape juice and red wine have beenassociated with potential cardiovascular benefits, including thereduction of platelet aggregation, modulation of eicosanoid synthesisand inhibition of low-density lipoprotein oxidation (Waterhouse andWalzem, 1997; Schramm et al., 1998; Frankel et al., 1995). Recently, ithas been suggested that any potential health benefits attributed tothese compounds may be affected by the degree of polymerization (Saitoet al. 1998).

Many plant polyphenols have antioxidant activity and have an inhibitoryeffect on mutagenesis and carcinogenesis. For example, U.S. Pat. No.5,554,645 and U.S. Pat. No. 5,712,305 disclose cocoa polyphenolextracts, particularly procyanidins, which have been shown to possesssignificant biological utility. International_Publication WO 97/36497(published Dec. 24, 1997) discloses that these extracts also function toreduce periodontal disease, arteriosclerosis and hypertension; inhibitLDL oxidation and DNA topoisomerase II; modulate cyclo-oxygenase,lipoxygenase, nitric oxide or NO-synthase, apoptosis and plateletaggregation; and possess anti-inflammatory, antigingivitis andantiperiodontis activity. Moreover, WO 97/36497 discloses thatpolyphenol oligomers 5-12 possess enhanced anti-cancer activity comparedto the other polyphenolic compounds isolated from cocoa. Thus,consumption of these higher oligomers in cocoa products may providesignificant health benefits.

As previously noted, the use of cocoa extracts or polyphenols derivedthere from as NO or NO-synthase modulators is described in InternationalPublication WO 97/36497. Nitric oxide has been shown to play a role inmany significant biological processes, such as neurotransmission, bloodclotting, blood pressure control, regulation of serum lipid levels,cardiovascular disease, cerebral circulation (vascular headache), and arole in the immune system's ability to kill tumor cells andintracellular parasites. P. Clarkson, et al., “Oral L-arginine ImprovesEndothelium dependent situation in Hypercholesterolemic Young Adults”,J. Clin, Innest. 97, No 8: 1989-1994 (April 1996), P. L. Feldman, etal., “The Surprising Life of Nitric Oxide”, Chem. & Eng. News, pp. 26-38(Dec. 20, 1993); S. H. Snyder, et al., “Biological Rules of NitricOxide”, Scientific American, pp. 68-77 (May 1992); P. Chowienczyk etal., “L-arginine: No More Than A Simple Amino Acid?”, Lancet, 350:901-30(Sep. 27, 1997); M. A. Wheeler, et al., “Efforts of Long Term OralL-Arginine on The Nitric Oxide Synthase Pathway in The Urine fromPatients with Interstitial Cystitis”, J. Urology 158:2045-2050 (December1997); A. Tenenbaum, “L-Arginine: Rediscovery in Progress”, Cardiology90:153-159 (1998); I. K. Mohan, et al., “Effort of L-arginine NitricOxide System On Chemical-Induced Diabetes Mellitus”, Free RadicalBiology & Medicine 25, No. 7: 757-765 (1998); S. Klahr, “The Role ofL-Arginine in Hypertension and Nephrotoxicity”, Pharmacology andTherapeutics, pp. 547-550 (1998); and R. H. Boger, et al., “DietaryL-arginine and L-Tocopheral Reduce Vascular Oxidation Stress andPreserve Endothelial Function in some Hypocholesteralemic Rabbits viaDifferent Mechanisms,” Arterosclerosis 141:31-43 (1998).

For example, health benefits from various foods have been suggested.Peanuts have been reported to be a source of resveratrol, the compoundfound in grapes and red wine that has been linked to reducedcardiovascular disease. A diet including walnuts has been found toresult in reduced serum lipid levels and blood pressure. See Sabate, J.et al., “Effects of Walnuts on Serum Lipid Levels And Blood Pressure inNormal Men”, New England J. Med. 328:603-607 (Mar. 4, 1993). It has alsobeen suggested that frequent consumption of nuts may offer protectionfrom coronary heart disease. See Sabate, J. et al., “Nuts: A NewProtective Food Against Coronary Heart Disease”, Lipidology 5:11-16(1994). Without wishing to be bound by any theory, a postulatedmechanism of action, among others, includes the presence of relativelyhigh levels of arginine in nuts which results in nitric oxideproduction, thereby causing relaxation of vascular smooth muscle. It isbelieved that L-arginine is a substrate for nitric oxide production vianitric oxide synthase.

Accordingly, products, such as confectioneries and cocoa-containingproducts (cocoa powders, chocolate liquors, or extracts thereof) havinga high cocoa polyphenol concentration, especially a high concentrationof cocoa polyphenol oligomers 5-12 would be desirable. It would also behighly desirable to provide products containing effective amounts ofboth polyphenols, particularly the cocoa procyanidin(s), and L-arginineto stimulate the production of nitric oxide and elicit the healthbenefits provided therefrom.

SUMMARY OF THE INVENTION

The invention relates to novel food products comprising at least onepolyphenol (i.e., cocoa and/or nut procyanidin) and L-arginine in acombined amount effective to induce a physiological increase in nitricoxide production in a mammal after ingesting the food product. Theprocyanidin may be synthetic or natural. In a preferred embodiment, thecocoa polyphenol and L-arginine are provided, respectively, by apolyphenol-containing component (e.g., or cocoa and/or cocoa powderand/or nut skin ingredient) and an L-arginine containing component(e.g., a nut meat). However, this invention also encompasses foodproducts in which cocoa and/or nut polyphenol and/or L-arginine is,either of which may be natural or synthetic, added directly to the foodproduct.

The food products of this invention provide health benefits to themammals ingesting the food products. A particularly advantageous healthbenefit is the reduction of blood pressure. Other health benefits mayinclude reduced cardiovascular disease, anti-cancer activity,anti-oxidant activity, treatment of renal disease, enhanced immunefunction, and improved cognitive function.

The cocoa polyphenols contained in the food products of this inventionare preferably cocoa polyphenol oligomers 2-18, and more preferablycocoa polyphenol oligomers 5-12.

Cocoa polyphenols, which contain procyanidins, are present in cocoabeans. They are obtained by solvent extraction of powdered unfermentedbeans as described in U.S. Pat. No. 5,554,645. They are also present inchocolate components prepared from cocoa beans.

Suitable cocoa procyanidin-containing ingredients include roasted cocoanibs, chocolate liquor, partially defatted cocoa solids, nonfat cocoasolids, cocoa powder milled from the cocoa solids, and mixtures thereof.Preferably, the ingredients are prepared from underfermented beans sincethese beans contain higher amounts of cocoa polyphenols including thecocoa procyanidins.

One particularly preferred food product of this invention areconfectioneries, most preferably chocolates, which include Standard ofIdentity and Non-Standard of Identity chocolates. The food products ofthis invention may also be non-chocolate food products. Preferablenon-chocolate food products include nut based products such as peanutbutter, peanut brittle and the like. Another preferable food product ofthis invention is a low fat food product prepared with defatted orpartially defatted nut meats.

The L-arginine may be derived from any available arginine source, e.g.,Arachis hypogaea (peanuts), Juglans regia (walnuts), Prunus amygdalus(almonds), Corylus avellana (hazelnuts), Glycine max (soy bean) and thelike. Also useful are Carya illinoensis (pecans), Amacardium occidentale(cashews), and Macadamia integrifolia, M. tetraphylla (macadamia nuts).It is known that the L-arginine content of nuts can vary according tothe nut's maturity and, in addition, certain cultivars may have higherlevels. Related species of each genera will also be useful herein.Peanuts generally have about 2-3 g of L-arginine per 100 g of nutmeat.L-arginine content of almonds is about 2-3 g per 100 g, of walnuts about2-4 g per 100 g, of hazelnuts about 1.5-2.5 g per 100 g, and of pecansand macademia nuts about 0.5-1.5 g per 100 g. The nut may be nut pieces,a nut skin, a nut paste, and/or a nut flour present in amounts whichprovide the desired amount of L-arginine, which will vary depending uponthe nut source.

The L-arginine-containing ingredient may also be a seed, a seed paste,and/or a seed flour. Suitable seeds include Helianthus annuus (sunflowerseeds), Sesamum indicum (sesame seeds), fenugreek seeds, Cucurbitaspp.(pumpkins seeds) and the like. Sunflower seeds, pumpkin seeds, andsesame seeds respectively contain about 1.5-3.0 g, about 3.5-6.0 g, andabout 2-3 g of L-arginine per 100 g.

Another source high in L-arginine is gelatin which contains about 5 g ofL-arginine per 100 g of gelatin.

The food product contains at least about 200 mg, preferably 300 mg, ofprocyanidins per 100 grams of product and at least about 0.9 g,preferably 1.2 g, more preferably 1.6 g of L-arginine per 100 grams offood product

The food product may contain polyphenols from a source other than cocoa,e.g., the polyphenols found in the skins of nuts such as those describedabove. Peanut skins contain about 17% procyanidins, and almond skinscontain up to 30% procyanidins. In a preferred embodiment, the nut skinsare used to the food product, e.g., the nougat of a chocolate candy.Polyphenols from fruits and vegetables may also be suitable for useherein. It is known that the skins of fruits such as apples and oranges,as well as grape seeds, are high in polyphenols.

Without being bound to theory, it is believed that the combination ofthe cocoa polyphenol(s) and L-arginine provides unexpectedly enhancedhealth benefits because of the positive polyphenol modulation of NOand/or NO-synthase in the presence of L-arginine, a substrate forNO-synthase. Thus, nitric oxide production is increased by thecombination of cocoa and/or nut polyphenol and L-arginine which resultsin improved health benefits derived from nitric oxide, e.g., theprevention of cardiovascular disease, reduced blood pressure,anti-cancer activity, and the like.

This invention is also related to a pharmaceutical compositioncomprising at least one cocoa and/or nut polyphenol(s), L-arginine, anda pharmaceutically acceptable carrier. The polyphenol(s) and L-arginineare present in a combined amount effective to induce a physiologicalincrease in nitric oxide production in a mammal ingesting thecomposition. The procyanidin(s) from the cocoa and/or the nut arepresent in an amount between 1 μg to about 10 g per unit dose. TheL-arginine is present in an amount of about 1 μg to about 10 g per unitdose. The cocoa polyphenol ingredient may be an extract of a cocoamaterial (beans, liquor, or powder, etc.) or may be a synthesizedderivative thereof, or may be synthesized polyphenol compound or mixtureof polyphenol compounds or derivatives thereof. Procyanidin extractedfrom nut skins are also suitable for use herein.

DETAILED DESCRIPTION OF THE INVENTION

The food product of this invention contains at least one cocoapolyphenol and optionally polyphenols from other sources as discussedabove. The cocoa polyphenol may be from any source, i.e., natural orsynthesized. Most preferably, the cocoa polyphenol is an oligomer.

The term “cocoa polyphenol” includes the procyanidins present in cocoabeans or a cocoa ingredients used in the production of chocolateconfectioneries, extracts of cocoa beans or a cocoa ingredientcomprising procyanidins, and synthesized derivatives thereof, andincludes synthesized cocoa polyphenol compounds or synthesized mixturesof cocoa polyphenol compounds, and derivatives thereof. The cocoa beansmay be fully fermented or underfermented.

The term “cocoa ingredient” refers to a cocoa solids-containing materialderived from shell-free cocoa nibs and includes chocolate liquor,partially or fully defatted cocoa solids (e.g., cake or powder,alkalized cocoa powder, or alkalized chocolate liquor and the like).

The term “chocolate liquor” refers to the dark brown fluid “liquor”formed by grinding a cocoa nib. The fluidity is due to the breakdown ofthe cell walls and the release of the cocoa butter during the processingresulting in a suspension of ground particles of cocoa solids suspendedin cocoa butter.

Partially defatted cocoa solids having a high cocoa polyphenol (CP)content, including a high cocoa procyanidin content, can be obtained byprocessing the cocoa beans directly to cocoa solids without a bean ornib roasting step. This method conserves the cocoa polyphenols becauseit omits the traditional roasting step. The method consists essentiallyof the steps of: a) heating the cocoa beans to an internal beantemperature just sufficient to reduce the moisture content to about 3%by weight and to loosen the cocoa shell; b) winnowing the cocoa nibsfrom the cocoa shells; c) screw pressing the cocoa nibs; and d)recovering the cocoa butter and partially defatted cocoa solids whichcontain cocoa polyphenols including cocoa procyanidins. Optionally, thecocoa beans are cleaned prior to the heating step, e.g., in an airfluidized bed density separator. The winnowing can also be carried outin the air fluidized bed density separator. Preferably, the cocoa beansare heated to an internal bean temperature of about 100° C. to about110° C., more preferably less than about 105° C., typically using ainfra red heating apparatus for about 3 to 4 minutes. If desired, thecocoa solids can be alkalized and/or milled to a cocoa powder.

The internal bean temperature (IBT) can be measured by filling aninsulated container such as a thermos bottle with beans (approximately80-100 beans). The insulated container is then appropriately sealed inorder to maintain the temperature of the sample therein. A thermometeris inserted into the bean-filled insulated container and the temperatureof the thermometer is equilibrated with respect to the beans in thethermos. The temperature reading is the IBT temperature of the beans.IBT can also be considered the equilibrium mass temperature of thebeans.

Cocoa beans can be divided into four categories based on their color:predominately brown (fully fermented), purple/brown, purple, and slaty(unfermented). Preferably, the cocoa solids are prepared fromunderfermented cocoa beans which have a higher cocoa polyphenol contentthan fermented beans. Underfermented beans include slaty cocoa beans,purple cocoa beans, mixtures of slaty and purple cocoa beans, mixturesof purple and brown cocoa beans, or mixture of slaty, purple, and browncocoa beans. More preferably, the cocoa beans are slaty and/or purplecocoa beans.

As discussed above, the cocoa polyphenol (CP) content, including thecocoa procyanidin content, of roasted cocoa nibs, chocolate liquor, andpartially defatted or nonfat cocoa solids is higher when they areprepared from cocoa beans or blends thereof which are underfermented,i.e., beans having a fermentation factor of 275 or less.

The “fermentation factor” is determined using a grading system forcharacterizing the fermentation of the cocoa beans. Slaty is designated1, purple is 2, purple/brown is 3, and brown is 4. The percentage ofbeans falling within each category is multiplied by the weighted number.Thus, the “fermentation factor” for a sample of 100% brown beans wouldbe 100×4 or 400, whereas for a 100% sample of purple beans it would be100×2 or 200. A sample of 50% slaty beans and 50% purple beans wouldhave a fermentation factor of 150 [(50×1)+(50×2)].

High CP chocolate liquor and/or high CP cocoa solids can be prepared by:a) roasting the selected cocoa beans (fermentation factor of 275 orless) to an internal bean temperature of 95° C. to 160° C.; b) winnowingthe cocoa nibs from the roasted cocoa beans; c) milling the cocoa nibsinto the chocolate liquor; and d) optionally recovering cocoa butter andpartially defatted cocoa solids from the chocolate liquor.Alternatively, the chocolate liquor and/or cocoa solids can be preparedby: a) heating the selected cocoa beans (fermentation factor of 275 orless) to an internal bean temperature of 95-135° C. to reduce themoisture content to about 3% by weight and to loosen the cocoa shellfrom the cocoa nibs; b) winnowing the cocoa nibs from the cocoa shells;c) roasting the cocoa nibs to an internal nib temperature of 95° C. to160° C.; d) milling the roasted nibs into the chocolate liquor; and (e)optionally recovering cocoa butter and partially defatted cocoa solidsfrom the chocolate liquor. Chocolate liquor and partially defatted cocoasolids containing at least 50,000 μg of total cocoa procyanidins and/orat least 5,000 μg of cocoa procyanidin pentamer per gram of nonfat cocoasolids can be prepared by the above processes.

An extract containing cocoa polyphenols including cocoa procyanidins canbe prepared by solvent extracting the partially defatted cocoa solids ornonfat cocoa solids prepared from the underfermented cocoa beans orcocoa nibs.

The partially defatted cocoa solids and/or cocoa polyphenol extracts canbe used in therapeutic compositions, optionally with a carrier or adiluent. The therapeutic compositions are useful as antineoplasticcompositions, antioxidants, antimicrobial agents, nitric oxide (NO) orNO-synthase modulators, cyclo-oxygenase modulators, lipoxygenasemodulators, and in vivo glucose modulators.

High CP food products may be prepared using the high CP roasted cocoanibs, high CP chocolate liquors, and/or high CP partially defatted ornonfat cocoa solids. The food products include pet food, dry cocoamixes, puddings, syrups, cookies, savory sauces, rice mixes, rice cakes,beverage mixes, beverages and the like. Preferably, the food productsare confectioneries, e.g., a dark chocolate or a milk chocolate. Theextract can also be used to prepare foods having high cocoa polyphenolcontents.

The health of a mammal can be improved by administering to the mammal acomposition containing cocoa and/or nut procyanidins or the above highCP cocoa components and/or nut components. In these compositions thetotal amount of the procyanidin oligomer(s) is at least 1 μg or greaterand the composition is administered daily over greater than 60 days.

Cocoa procyanidins may be structurally represented as oligomers ofmonomer A, having the formula A_(n), where n is 2-18, where, A has theformula:

and R is 3-(α)-OH, 3-(β)-OH, 3-(α)-O-saccharide, 3-(β)-O-saccharide;bonding between adjacent monomers takes place at positions 4, 6 or 8;with the proviso that X, Y and Z are selected from the group consistingof A, hydrogen, and a saccharide; a bond to a monomer in position 4 hasalpha or beta stereochemistry, as to at least one terminal monomer; andbonding of the adjacent monomer thereto is at position 4. OptionallyY=Z=hydrogen; and salts thereof; wherein the saccharide moiety isderived from a mono- or di-saccharide.

The term “oligomer”, as used herein, refers to any compound having theabove formula, presented above, wherein n is 2 through 18, andpreferably, wherein n is 5-12. When n is 2, the oligomer is termed a“dimer”; when n is 3, the oligomer is termed a “trimer”; when n is 4,the oligomer is termed a “tetramer”; when n is 5, the oligomer is termeda “pentamer”; and similar recitations may be designated for oligomershaving n up to and including 18 and higher, such that when n is 18, theoligomer is termed an “octadecamer”.

Synthesized derivatives of the cocoa polyphenols include compounds,according to the structure A_(n), above, wherein R may be3-(α)-O-saccharide, 3-(β)-O-saccharide, 3-(α)-O—C(O)—R¹, or3-(β)-O—C(O)—R¹ wherein the saccharide moiety may be derived from amono- or di-saccharide selected from the group consisting of glucose,galactose, xylose, rhamnose and arabinose; wherein the saccharide moietyof any or all of R, X, Y, and Z may be optionally substituted at anyposition with a phenolic moiety via an ester bond; wherein the phenolicmoiety may be selected from the group consisting of caffeic, cinnamic,coumaric, ferulic, gallic, hydroxybenzoic and sinapic acids; and whereinR¹ is an aryl or heteroaryl moiety optionally substituted with at leastone hydroxyl moiety. The substituted aryl or heteroaryl group of R¹ maypreferably contain a substitution pattern corresponding to thesubstituted phenolic groups of caffeic, cinnamic, coumaric, ferulic,gallic, hydroxybenzoic or sinapic acids.

The polyphenol oligomers may be prepared by

(a) protecting each phenolic hydroxyl group of a first and a secondpolyphenol monomer with a protecting group to produce a first and secondprotected polyphenol monomer;

(b) functionalizing the 4-position of the first protected polyphenolmonomer to produce a functionalized protected polyphenol monomer havingthe formula:

wherein:

-   -   c is an integer from 1 to 3;    -   d is an integer from 1 to 4;    -   y is an integer from 2 to 6;    -   R is a protecting group; and    -   R* is H or OH;

(c) coupling the second protected polyphenol monomer with thefunctionalized protected polyphenol monomer to produce a protectedpolyphenol dimer as the polyphenol oligomer;

(d) optionally repeating the functionalization and coupling steps toform the polyphenol oligomer having n monomeric units, wherein n is aninteger from 3 to 18; preferably 5-12; and

(e) removing the protecting groups from the phenolic hydroxyl groups.

The preferred protected polyphenol monomer is a brominated protectedepicatechin or brominated protected catechin, more preferably an8-bromo-epicatechin or an 8-bromo-catechin.

In the above process, the 4-position of the protected polyphenol monomermay be oxidatively functionalized using a quinone oxidizing agent in thepresence of a diol, e.g., ethylene glycol when y is 2.

The above process may further comprise the step of forming a derivativeof the polyphenol oligomer by esterifying the polyphenol oligomer at the3-position of at least one monomeric unit to produce an esterifiedpolyphenol oligomer. The ester group may be selected from the groupconsisting of —OC(O)-aryl, —OC(O)-substituted aryl, —OC(O)-styryl, andOC(O)-substituted styryl, where the substituted aryl or substitutedstyryl contains at least one substituent selected from the groupconsisting of halo, hydroxyl, nitro, cyano, amino, thiol,methylenedioxy, dihalomethylenedioxy, a C₁-C₆ alkyl, a C₁-C₆ alkoxy, aC₁-C₆ haloalkyl, a C₁-C₆ haloalkoxy, a C₃-C₈ cycloalkyl and a C₃-C₈cycloalkoxy. Preferably, the 3-position of at least one monomeric unitis converted to a derivative group derived from an acid selected fromthe group consisting of caffeic, cinnamic, coumaric, ferulic, gallic,hydroxybenzoic and sinapic acids.

The above process may further comprise the step of forming a derivativeof the polyphenol oligomer by glycosylating the polyphenol oligomer atthe 3-position of at least one monomeric unit to produce a glycosylatedpolyphenol oligomer. Preferably, the 3-position of at least onemonomeric unit is converted to a derivative group selected from thegroup consisting of —O-glycoside or an —O-substituted glycoside whereinthe substituted glycoside is substituted by —C(O)-aryl,—C(O)-substituted aryl, —C(O)-styryl, or —C(O)-substituted styryl. Thesubstituted aryl or substituted styryl may contain substituents selectedfrom the group consisting of halo, hydroxyl, nitro, cyano, amino, thiol,methylenedioxy, dihalomethylenedioxy, a C₁-C₆ alkyl, a C₁-C₆ alkoxy, aC₁-C₆ haloalkyl, a C₁-C₆ haloalkoxy, a C₃-C₈ cycloalkyl and a C₃-C₈cycloalkoxy. Preferably, the glycoside is selected from the groupconsisting of glucose, galactose, xylose, rhamnose, and arabinose.

The food products of this invention may contain one or more of the cocoapolyphenol monomers, oligomers 2-18, or derivatives thereof. Preferably,the food products of this invention contain mixtures of cocoa polyphenololigomers 2-18, or derivatives thereof; more preferably, the foodproducts contain mixtures of cocoa polyphenol oligomers 5-12, orderivatives thereof.

The food products of this invention include products meant for ingestionby humans and other mammals, e.g. dogs, cats, horses and the like. Thefood products of this invention can be ingested for nourishment,pleasure, or medical or veterinary purposes.

A preferred food product is a confectionery, a baked product, acondiment, a granola bar, meal replacement bar, a syrup, a powderbeverage mix, a beverage, and the like. More preferably, the foodproduct of this invention is a chocolate confectionery containing nuts,e.g., peanuts, walnuts, almonds, hazelnuts, nuts, and the like. The nutmeats can be in any form, e.g., whole nuts, chopped nuts, ground nuts,nut pastes, or the like. The preferred non-chocolate food productsinclude peanut butter, peanut brittle and the like. Such non-chocolatefood products may contain cocoa ingredients, particularly cocoapolyphenol—containing cocoa ingredients, but would not be considered achocolate product by one of ordinary skill in the art, e.g., peanutbutter containing a relatively small percentage of cocoa powder havinghigh concentrations of cocoa polyphenols.

Chocolate used in foods in the United States is subject to a standard ofidentity (SOI) established by the U.S. Food and Drug Administrationunder the Federal Food, Drug and Cosmetic Act that sets out therequisite ingredients, and proportions thereof, of a confection topermit labeling of the confection as a “chocolate.”

The most popular chocolate or chocolate candy consumed in the UnitedStates is in the form of sweet chocolate or milk chocolate. Chocolate isessentially a mixture of cocoa solids suspended in fat. Milk chocolateis a confection which contains non-fat milk solids, milk fat, chocolateliquor, a nutritive carbohydrate sweetener, cocoa butter and may includea variety of other ingredients such as emulsifying agents, flavoringsand other additives. Sweet chocolate contains higher amounts ofchocolate liquor, but lower amounts of milk solids than milk chocolate.Semi-sweet chocolate requires at least 35% by weight chocolate liquorand is otherwise similar in definition to sweet chocolate. Darkchocolate generally contains only chocolate liquor, a nutritivecarbohydrate sweetener, and cocoa butter, and is by definition either asweet chocolate or a semi-sweet chocolate. Buttermilk chocolate and skimmilk chocolate differ from milk chocolate in that the milk fat comesfrom various forms of sweet cream, buttermilk, and skim milk,respectively. Skim milk requires the total amount of milk fat to belimited to less than the minimum for milk chocolate. Mixed dairy productchocolates differ from milk chocolate in that the milk solid includesany or all of the milk solids listed for milk chocolate, buttermilkchocolate, or skim milk chocolate. White chocolate differs from milkchocolate in that it contains no non-fat cocoa solids. Heat stablechocolates are also useful herein.

Non-standardized chocolates are those chocolates which have compositionswhich fall outside the specified ranges of the standardized chocolates.Chocolates are classified as “non-standardized” chocolates when aspecified ingredient is replaced, either partially or completely, suchas when the cocoa butter is replaced with vegetable oils or fats. Anyadditions or deletions to a chocolate recipe made outside the US FDAStandards of Identity for chocolate will prohibit use of the term“chocolate” to describe the confectionery. However, as used herein, theterm “chocolate” or “chocolate product” refers to any standard ofidentity or non-standard of identity chocolate product.

Chocolate may take the form of solid pieces of chocolate, such as barsor novelty shapes. Chocolate may also be incorporated as an ingredientin other more complex confections where chocolate is combined with andgenerally coats other foods inclusions such as caramel, peanut butter,nougat, fruit pieces, nuts, wafers, ice cream or the like. These foodsare characterized as microbiologically shelf-stable at 65°-85° F.(18-29° C.) under normal atmospheric conditions.

The term “carbohydrate” refers to nutritive carbohydrate sweeteners,with varying degrees of sweetness intensity and may be any of thosetypically used and include, but are not limited to, sucrose, (e.g., fromcane or beet), dextrose, fructose, lactose, maltose, glucose syrupsolids, corn syrup solids, invert sugar, hydrolyzed lactose, honey,maple sugar, brown sugar, molasses and the like.

The chocolate food products may additionally contain other ingredientssuch as non-fat milk solids, non-fat cocoa solids (cocoa powder), sugarsubstitutes, natural and artificial flavors (e.g., spices, coffee, salt,etc. as well as mixtures of these), proteins, and the like.

The food products of this invention also include L-arginine. AnyL-arginine source may be used, i.e., synthetic or natural. Particularlypreferred L-arginine sources include soy beans and nut meats such aspeanuts, walnuts, almonds, hazelnuts and the like. Defatted andpartially defatted nut meats may also be used to enhance the L-arginineconcentration. Partially or fully defatted ground nut meats are referredto as nut flours.

In addition to the physiological activities known to be elicited bycocoa procyanidins or compositions containing cocoa procyanidins, thecombination of L-arginine with the cocoa procyanidins produces bettereffect, as shown by the increased nitric oxide production.

One embodiment of a synergistic effect on NO and/or NO-synthasemodulation, for example follows. Many foods contain appreciable amountsof L-arginine, but not necessarily cocoa polyphenols. Given thatL-arginine is a substrate for NO-synthase, and NO dependentvasodilatation is significantly improved in hypercholesterolemic animalsreceiving L-arginine supplementation (See Cooke et al., Circulation83:1057-1062, 1991) and that cocoa polyphenols can modulate NO levels, asynergistic improvement in endothelium dependent vasodilatation isexpected. L-arginine levels of 1.0 to 1.1 g/100 g have been reported inunsweetened cocoa powder. From this basis, other sources of L-arginineare incorporated into the food products to provide for maximal benefitrelated to NO and NO-synthase modulation. In a particularly preferredembodiment, the cocoa and/or nut polyphenols and L-arginine are presentin amounts effective to provide the above described synergistic benefit,e.g., about 1 mg to about 10 g per unit dose, preferably about 25 mg to3 g of procyanidins. The products of the invention may be used forarresting cancer cell growth in mammals, for reducing hypertension inmammals, treating inflammatory bowel disease, for inhibiting bacterialgrowth in mammals, for preventing or reducing otherosclerosis orrestenosis, for modulating platelet aggregation, for modulatingapoptosis, as an antioxidant, specifically for preventing oxidation ofLDL in mammals, for modulating cyclo-oxygenase and/or lipoxygenase, formodulating or stimulating nitric oxide (NO) production or nitric oxide(NO) synthase in mammals, for treating nitric oxide (NO) affectedhypercholesterolemic in a mammal, for modulating in vivo glucos, forinhibiting topoisomerase II, for inducing INOS in mammalian monocyteand/or macrophage, as well as an antimicrobial, antineoplastic,anti-gingivitis or anti-periodontitis agent.

Using the food products and pharmaceutical compositions of thisinvention containing cocoa and/or nut polyphenols and L-arginine, novelmethods of improving the health of a mammal, particularly a human, maybe practiced.

A preferred embodiment of the invention is a method of improving thehealth of a mammal by administering an effective amount of the foodproduct or pharmaceutical composition containing cocoa and/orpolyphenols and L-arginine to the mammal each day for an effectiveperiod of time. Depending on the condition treated, the effective periodof time may vary from almost instantaneous to a period greater thansixty days. In one aspect, the mammal's health is improved by ingestingan edible composition containing cocoa polyphenols and L-arginine eachday for a period of time greater than five days to a period of timegreater than sixty days.

The polyphenols used in this invention modulate nitric oxide (NO) andNO-synthase. The arginine acts as a substrate for NO-synthase. Thecombined amount of cocoa polyphenol and L-arginine is effective toelicit a physiological response in a mammal receiving the food product.The physiological response is increased in nitric oxide production overthat which would be obtained by the administration of the cocoapolyphenol or L-arginine alone. It is believed that this enhanced nitricoxide production results in the aforementioned health benefitsassociated with nitric oxide production.

The food products and pharmaceutical compositions of this invention areuseful, for example, in modulating vasodilation, and are further usefulwith respect to modulating blood pressure or addressing coronaryconditions, and migraine headache conditions. The responses elicitedupon administration of the compositions of this invention includelowering hypertension and dilating blood vessels.

The novel food products of this invention can be readily prepared bythose of ordinary skill in the art using the teachings set forth herein.

The cocoa ingredients can be prepared from cocoa beans having afermentation factor of less than 300 and/or from cocoa beans having afermentation factor of 300 or greater. Alkalized chocolate ingredientsprepared from cocoa beans having a fermentation factor of 300 or greatercan be used in combination with cocoa ingredients prepared from cocoabeans having fermentation factor of less than 300.

The cocoa procyanidin content of chocolate-based food products can beconserved by protecting the carbohydrate ingredient(s) and/or the milkingredient(s) during formulation of the food product. The ingredient(s)are protected before adding the chocolate ingredient(s). At least oneprotective ingredient selected from the group consisting of a fat, anemulsifying agent, an antioxidant, a flavorant, and mixtures thereof isadded to the carbohydrate ingredient(s) and/or milk ingredient(s) toform a first mixture. The first mixture is combined with the chocolateingredient(s) to form a second mixture. The food product is formed fromthe second mixture. The food product may be a confectionery or a dietsupplement. The confectionery may be a dark or milk chocolate.Optionally, the carbohydrate(s) and/or milk ingredient(s) are milled toreduce the particle size prior to mixing with the protective ingredient.The chocolate ingredient(s) may also be milled prior to being combinedwith the first mixture of protected carbohydrate and/or milkingredients. Preferred fats for use as pretreatment ingredients arecocoa butter and/or a chocolate liquor which contains cocoa butter andwhich is prepared from cocoa beans having a fermentation factor of 300or greater. Preferred emulsifying agents include lecithin and orfractionated lecithin. Suitable antioxidants include tannins, quinones,polyhydroxy compounds, phospholipids, tocol compounds, and/orderivatives thereof. Suitable flavoring agents include vanillin, spices,and/or naturally expressed citrus oils or spice oils. The first mixture,the chocolate ingredient(s), and/or the second mixture can be conched.The chocolate is conched at about 50 to about 65° C. A secondemulsifying agent can be added during or after conching. This secondemulsifying agent may be lecithin, sucrose polyeruiate, ammoniumphosphatide, polyglycerol, polyricinoleate, phosphated mono- anddi-glycosides/deactyl tartaric acid esters of monoglycerides, andfractionated lecithin. Food products prepared with the protectedcarbohydrate(s) and/or milk ingredient(s) contain at least 10 to 20% byweight more cocoa procyanidins than a food product prepared by a processthat does not include pretreatment of the carbohydrate ingredient(s)and/or milk ingredient(s).

The addition of L-arginine can be made to the food product by adding anamount of nut meat, e.g., peanuts sufficient to provide fits desiredconcentration of L-arginine.

As previously noted, a particularly preferred food product is achocolate confectionery. The chocolate in the chocolate confectionerycontains a relatively high concentration of cocoa polyphenols. In thisembodiment, the chocolate comprises at least 3,600 μg, preferably atleast 4,000 μg, preferably at least 4,500 μg, more preferably at least5,000 μg, and most preferably at least 5,500 μg cocoa procyanidins pergram of chocolate, based on the total amount of nonfat cocoa solids inthe product. According to one preferred embodiment, the chocolatecontains at least 6,000 μg, preferably at least 6,500 μg, morepreferably at least 7,000 μg, and most preferably at least 8,000 μg ofcocoa procyanidins per gram, and even more preferably 10,000 based onthe nonfat cocoa solids in the product.

Another embodiment relates to a chocolate food product comprising achocolate having at least 200 μg, preferably at least 225 μg, morepreferably at least 275 μg, and most preferably at least 300 μg cocoaprocyanidin pentamer per gram, based on the total amount of nonfat cocoasolids in the chocolate food product. Preferably, the chocolate containsat least 325 μg, preferably at least 350 μg, more preferably at least400 μg, and most preferably at least 450 μg cocoa procyanidin pentamerper gram, based on the total amount of nonfat cocoa solids in thechocolate food product.

Yet another embodiment, relates to a milk chocolate confectionery whichhas at least 1,000 μg, preferably at least 1,250 μg, more preferably atleast 1,500 μg, and most preferably at least 2,000 μg cocoa polyphenolsper gram, based on the total amount of nonfat cocoa solids in the milkchocolate product. In the preferred embodiment, the milk chocolatecontains at least 2,500 μg, preferably at least 3,000 μg, morepreferably at least 4,000 μg, and most preferably at least 5,000 μgcocoa procyanidins per gram, based on the total amount of nonfat cocoasolids in the milk chocolate product.

In another embodiment, the food product is a milk chocolate which has atleast 85 μg, preferably at least 90 μg, more preferably at least 100 μg,and most preferably at least 125 μg cocoa procyanidin pentamer per gram,based on the total amount of nonfat cocoa solids in the milk chocolateproduct. In a preferred embodiment, the milk chocolate contains at least150 μg, preferably at least 175 μg, more preferably at least 200 μg, andmost preferably at least 250 μg cocoa procyanidin pentamer per gram,based on the total amount of nonfat cocoa solids in the milk chocolateproduct.

The non-chocolate food products will contain at least 1 μg, preferablyat least 5 μg, more preferably at least 10 μg, more preferably at least25 μg, and most preferably at least 50 μg of cocoa procyanidins. Ifdesired, the non-chocolate food products can contain much higher levelsof cocoa procyanidins compared to those found in the above-describedchocolate food products.

The amount of L-arginine in the food products can vary. Typically, cocoacontains between 1 to 1.1 grams of L-arginine per 100 grams of partiallydefatted cocoa solids. It can range from 0.8 to 1.5 per 100 grams ofcocoa. The chocolate food products of this invention contain L-argininein an amount greater than that which naturally occurs in the cocoaingredients. Knowing the amount of cocoa ingredients and L-arginine usedin the food product, one of ordinary skill in the art can readilydetermine the total amount of L-arginine in the final product.

The food product will generally contain at least 1 μg, preferably atleast 10 μg, or at least 100 μg, even more preferably at least 1000 μg,or 5,000 or 10,000 μg, and most preferably at least 20,000, 50,000 or100,000 μg of L-arginine per gram of food product.

As previously noted, this invention is also directed to a pharmaceuticalcomposition comprising at least one cocoa polyphenol, L-arginine and apharmaceutically acceptable composition. Inclusion of L-arginine inamounts ranging from about 1 μg to about 10 grams per unit dose may bereadily performed by one of ordinary skill in the art. Thepharmaceutical compositions of this invention are useful for treatingmammals in need of increased nitric oxide production and the benefitsthat flow therefrom, such as reduced blood pressure.

Test Procedures

The following procedures can be used for quantifying the amount ofprocyanidins and L-arginine in the various examples.

Method A was used for quantification of the cocoa procyanidin amounts(total and pentamer) reported in Examples 1 to Example 3.

Method B should be used for quantification of the cocoa and nutprocyanidin amounts (total and pentamer) in the food products and foodingredients of Examples 4 to 10. Method B was used for quantification ofthe cocoa procyanidin content of the purified cocoa procyanidinoligomers reported in Example 14 and used in Examples 17-19.

Method C should be used for extracting and identifying nut procyanidins.

Determination of Procyanidin

Method A

Cocoa polyphenol extracts are prepared by grinding a 6-7 g sample usinga Tekmar A-10 Analytical Mill for 5 minutes, or, in the case ofchocolate liquors, from 6-7 g of chocolate liquor sample withoutadditional grinding. The sample is then transferred to a 50 mLpolypropylene centrifuge tube, approximately 35 mL of hexane is added,and sample is shaken vigorously for 1 minute. Sample is spun at 3000 RPMfor 10 minutes using an International Equipment Company IECPR-7000Centrifuge. After decanting the hexane layer, the fat extraction processis repeated two more times. Approximately 1 g of the defatted materialis weighed into a 15 mL polypropylene centrifuge tube and 5 mL of a 70%acetone:29.5% water:0.5% acetic acid solution is added. The sample isvortexed for about 30 seconds using a Scientific Industries Vortex Genie2 and spun at 300 RPM for 10 minutes in the IECPR-7000 Centrifuge. Theliquor is then filtered into a 1 ml hypovial through a Millex-HV 0.45 gfilter.

Cocoa polyphenol extracts are analyzed by a Hewlett Packard 1090 SeriesII HPLC system equipped with a HP model 1046A Programmable Fluorescencedetector and Diode Array detector. Separations are effected at 37° C. ona 5 g Supelco Supelcosil LC-Si column (250×4.6 mm) connected to aSupelco Supelguard LC-Si 5 μm guard column (20×2.1 mm). Procyanidins areeluted by linear gradient under the following conditions: (time % A, %B, % C); (0, 82, 14, 4), (30, 67.6, 28.4, 4), (60, 46, 50, 4), (65, 10,86, 4), followed by a 5 minute re-equilibration. Mobile phasecomposition is A=dichloromethane, B=methanol, and C=acetic acid:water ata volume ratio of 1:1. A flow rate of 1 mL/min is used. Components aredetected by fluorescence, where λ_(ex)=276 nm and λ_(em)=316 nm, or byUV at 280 nm. Epicatechin is used as an external standard.

HPLC conditions:

250×4.6 mm Supelco Supelcosil LC-Si column (5 μm) 20×2.1 mm SupelcoLC-Si (5 μm) guard column

Detectors: Photodiode Array at 280 nm

Fluorescence λ_(ex)=276 nm; λ_(em)=316 nm

Flow rate: 1 mL/min

Column temperature: 37° C.

Acetic Acid Gradient CH₂Cl₂ Methanol Water (1:1) 0 82 14 4 30 67.6 28.44 60 46 50 4 65 10 86 4

Method B

In this method the monomeric and oligomeric cocoa and nut procyanidinsare quantitated using a normal-phase high performance liquidchromatography (HPLC) method with fluorescence detection (FLD) insteadof UV detection at 280 nm.

The normal-phase HPLC method reported by Hammerstone et al.,“Identification of Procyanidins in Cocoa (Theobroma cacao) and ChocolateUsing High Performance Liquid Chromatography/Mass Spectrometry”, J.Agric. Food Chems. 47, 2:490-496(Jan. 14, 1999) was used for theseparation and quantification of oligomers up to the decamer.

Procyanidin standards through decamers were obtained by extraction fromcocoa beans, enrichment by Sephadex LH-20 gel permeation chromatography,and final purification by preparative normal-phase HPLC. The purity ofeach oligomeric fraction was assessed using HPLC coupled to massspectrometry.

A composite standard was then prepared and calibration curves weregenerated for each oligomeric class using a quadratic fit of area sumversus concentration.

Cocoa beans were provided by the Almirante Center for Cocoa Studies inItajuipe, Brazil.

The reference compounds are (−)-epicatechin (Sigma Chemical, St. Louis)and purified oligomers from Brazilian cocoa beans.

The cocoa procyanidins are extracted by grinding the fresh seeds in ahigh-speed laboratory mill with liquid nitrogen until the particle sizeis reduced to approximately 90 μm. Lipids are removed from 220 g of theground seeds by extracting three times with 1000 mL of hexane. Thelipid-free solids are air dried to yield approximately 100 g of fat freematerial. A fraction containing procyanidins is obtained by extractingwith 1000 mL of 70% by volume acetone in water. The suspension iscentrifuged for 10 minutes at 1500×g. The acetone layer is decantedthrough a funnel with glass wool. The aqueous acetone is thenre-extracted with hexane (˜75 mL) to remove residual lipids. The hexanelayer is discarded and the aqueous acetone is rotary evaporated underpartial vacuum at 40° C. to a final volume of 200 mL. The aqueousextract is freeze-dried to yield approximately 19 g of acetone extractmaterial.

For the gel permeation chromatography approximately 2 g of the acetoneextract is suspended in 10 mL of 70% aqueous methanol and centrifuged at1500×g. The supernatant is semi-purified on a Sephadex LH-20 column(70×3 cm) which has previously been equilibrated with methanol at a flowrate of 3.5 mL/min. Two and a half hours after sample loading, fractionsare collected every 20 minutes and analyzed by HPLC for theobromine andcaffeine (Clapperton et al., “Polyphenols and Cocoa Flavour”,Proceedings, 16^(th) International Conference of group Polyphenols,Lisbon, Portugal, Grouppe Polyphenols, Norbonne France, Tome II:112-1151992). Once the theobromine and caffeine are eluted off the column (˜3.5hours), the remaining eluate is collected for an additional 4.5 hoursand rotary evaporated under partial vacuum at 40° C. to remove theorganic solvent. Then the extract is suspended in water andfreeze-dried.

The cocoa procyanidin oligomers are purified by preparative normal-phaseHPLC. Approximately 0.7 g of semi-purified acetone extract is dissolvedin 7 mL of acetone:water:acetic acid in a ratio by volume of70:29.5:0.5, respectively. Separations were effected at ambienttemperature using a 5μ Supelcosil LC-Si 100 Å (50×2 cm). Procyanidinswere eluted by a linear gradient under the conditions shown in the Tablebelow. Separations of oligomers are monitored by UV at 280 nm andfractions are collected at the valleys between the peaks correspondingto oligomers. Fractions with equal retention times from severalpreparative separations are combined, rotary evaporated under partialvacuum and freeze-dried.

Gradient Profile for Preparative Normal Phase HPLC

Methylene Chloride:acetic Methanol:acetic Time acid:water acid:waterFlow rate (min) (96:2.2 v/v) (96:2.2 v/v) (mL/min) 0 92.5%  7.5% 10 1092.5%  7.5% 40 30 91.5%  8.5% 40 145 78.0% 22.0% 40 150 14.0% 86.0% 40155 14.0% 86.0% 50 180   0%  100% 50

For the mass spectrometry analysis of the partially purified cocoaprocyanidin oligomers, purified fractions are analyzed by HPLC/massspectrometry (MS) using the parameters described by Lazarus et al. “HighPerformance Liquid Chromatography/mass Spectrometry Analysis ofProanthocyanidins in Food Stuffs”, J. Agric. Food Chem. (submitted in1998). Purities of each fraction are determined by peak area using UVdetection at 280 nm in combination with comparing the ratio of ionabundances between each oligomeric class. Composite standard stocksolutions are made using commercially available (−)-epicatechin for themonomer and the purified oligomers for dimers through decamers. Theoligomeric profile of the composite standard stock solution is shown inthe following Table.

Oligomeric Profile of Composite Standard

Cocoa Contribution Procyanidins (% by weight) Monomer 9.82 Dimer 13.25Trimer 9.85 Tetramer 10.49 Pentamer 10.51 Hexamer 12.68 Heptamer 7.98Octamer 8.44 Nonamer 11.56 Decamer 5.42

Stock solutions are made at the following concentrations: 20 mg/mL, 10mg/mL, 5 mg/mL, 2 mg/mL, 1 mg/mL and 0.4 mg/mL.

Chocolate liquors and chocolate samples are extracted as above onlyusing (approximately 8 g of sample) 45 mL of hexane. Approximately 1 gof defatted material is extracted as above with 5 mL ofacetone:water:acetic acid. The solids are pellitized by centrifuging for10 mins at 1500×g. Then the supernatant is filtered through a 0.45nicron nylon filter into an HPLC vial for injection. All defattedsamples are weighted, extracted and injected in duplicate. The fatcomposition of cocoa liquors and chocolates is determined using the AOACOfficial Method 920.177. A slight modification to the sample size isneeded which incorporates the use of 1 g for the chocolate samples and0.5 g for the liquor samples. High performance liquid chromatographicanalysis of cocoa procyanidins are performed using a HP 1100 Series HPLC(Hewlett Packard, Palo Alto, Calif.) equipped with an auto-injector,quaternary HPLC pump, column heater, fluorescence detector and HPChemStation for data collection and manipulation. Fluorescence detectionis recorded at excitation wavelength 276 nm and emission wavelength 316nm. Normal phase separations of the procyanidin oligomers are performedusing a Phenomenex (Torrance, Calif.) 5μ Lichrosphere silica column(25×4.6 mm) at 37° C. with a 5 μL injection volume. The ternary mobilephase consists of A) dichloromethane, B) methanol and C) acetic acid andwater (1:1 v/v). Separations are effected by a series of lineargradients of B into A with a constant 4% C at a flow rate of 1 mL/min asfollows: elution starting with 14% B in A; 14-28.4% B in A, 0-30 mins;28.4-39.2% B in A, 30-45 min; 39.2-86% B in A, 45-50 min. The columnsare re-equilibrated between injections with the equivalent of 25 mL (10column volumes) of the initial mobile phase.

For quantification of cocoa procyanidins in chocolate liquors andchocolates, calibration curves are made from the stock solutions using aquadratic fit for the relationship of area sum versus concentration forthe peaks corresponding to each oligomeric class.

Method C

This method is used to determine the type of procyanidins in nuts.Monomeric and oligomeric procyanidins present in nuts are separated bydegree of polymerization and identified using a modified normal-phasehigh performance liquid chromatography (HPLC) method coupled withon-line mass spectrometry (MS) analysis using an atmospheric pressureionization electrospray (API-ES) chamber. Raw peanuts are provided byM&M/MARS (Hackettstown, N.J.). Raw almonds were provided by the AlmondBoard of California (Modesto, Calif.).

The standards used are (−)-epicatechin and (+)-catechin. (SigmaChemical, St. Louis, Mo.).

Solid phase extraction (SPE) columns, (Supelcosil Envi-18 20 mL columnsfrom Supelco, Inc., Bellafonte, Pa.) are rinsed with 3×5 mL of methanoland then conditioned with 3×5 mL of water prior to sample loading. Afterthe appropriate sample loading and rinse procedures, the columns aredried under vacuum for 1-2 min. The SPE column is then soaked in 10 mLof acetone, water and acetic acid in a ratio by volume of 70:29.5:0.5,respectively, for 1 minute before the procyanidins are eluted off thecolumn. To extract the procyanidins from peanut skins, approximately 3.5g of peanut skins are ground in a laboratory mill before being extractedin 25 mL of acetone, water and acetic acid in a ratio by volume of70:29.5:0.5, respectively. The suspension is centrifuged for 10 minutesat 1500×g and the supernatant decanted. Twenty milliliters of water isadded to the supernatant before the organic solvent is removed by rotaryevaporation under partial vacuum at 45° C. to yield approximately 22 mLof aqueous extract. The aqueous extract (22 mL) is loaded onto thepreconditioned SPE column and rinsed with 40 mL of water. Then theprocyanidins are eluted as above. To extract the procyanidins frompeanut nutmeat, the nutmeat is frozen in liquid nitrogen and then groundinto a powder in a laboratory mill. The nutmeat powder (˜10 g) isextracted three times with 45 mL of hexane to remove lipids. One gram ofthe resultant defatted nutmeat is extracted with 5 mL of acetone, waterand acetic acid in a ratio by volume of 70:29.5:0.5, respectively.

With the almond seedcoat, approximately 24 g of seedcoat are removedfrom the raw almonds using a razor blade. The seedcoat is then defattedtwice with 135 mL of hexane and centrifuged for 10 minutes at 1500×g toyield approximately 14.6 g of defatted material. The defatted seedcoatis extracted with 90 mL of acetone, water and acetic acid in a ratio byvolume of 70:29.5:0.5, respectively. Thirty milliliters of water areadded to the supernatant and the resulting acidified aqueous acetone isrotary evaporated under partial vacuum at 45° C. to a final volume of 50mL. The aqueous solution is loaded onto the preconditioned SPE column,rinsed with approximately 10 mL of water and the procyanidins eluted asabove.

HPLC/MS analyses of the extracts are performed using a HP 1100 SeriesHPLC as described in Method B above and interfaced to a HP Series 1100mass selective detector (model G1946A) equipped with an API-ESionization chamber. The buffering reagent is added via a tee in theeluant stream of the HPLC just prior to the mass spectrometer anddelivered with a HP 1100 series HPLC pump bypassing the degasser.Conditions for analysis in the negative ion mode include ˜0.75M ammoniumhydroxide as the buffering reagent at a flow rate of 0.04 mL/min, acapillary voltage of 3 kV, the fragmentor at 75 V, a nebulizing pressureof 25 psig, and drying gas temperature at 350° C. Data are collected ona HP ChemStation using both scan mode and selected ion monitoring.Spectra are scanned over a mass range of m/z 100-3000 at 1.96 s percycle.

The mass spectral data of almond seedcoat ions indicates the presence ofsingly linked procyanidin oligomers through heptamers, whereas the massspectral data for peanut skins indicates both singly and doubly linkedoligomers through octamers. No procyanidins are detected in the peanutmeat.

Determination of L-Arginine Content

The L-arginine is determined using the procedure reported in AOACOfficial method 982.30, AOAC Official Methods of Analysis 91995),Vitamins and Other Nutrients, Chapter 45, p. 59-61. The sample is acidhydrolyzed and each of 3 hydrolysates is analyzed using parametersoptimal for the amino acid analyzer being used. A standard L-argininesolution is used to calibrate the analyzer at least every 24 hours. Thenitrogen is determined by AOAC Official Method 955.04 C, 920.39 A,976.05 A, or other appropriate Kjidahl method. The uncorrected g/16 g Nis computed according to:

g L-arginine (uncorrected 16 g sample N=(n moles L-arginine×initialsample volume (mL)×MW L-arginine)/(volume sample injected (mL)×sampleweight (g)×% N for sample×6.25×10⁵)

The acid hydrolysis is carried out by placing about 0.1 g (weigh to 0.1mg accuracy) sample in hydrolysis tube, adding 10 mL 6N Hl, and mixing.The mixture is frozen in a dry ice-alcohol bath. A vacuum of <50 u isdrawn and held for 1 minute and the tube was sealed under vacuum. Thesample is hydrolyzed for 24 hours at 110+1. The tube is cooled andopened. The hydrolysate is filtered through Whitman No 1 paper. The tubeis rinsed three times with water and each rinse is filtered. Thefiltrate is dried at 65° under vacuum. The dry hydrolysate is dissolvedin a volume of buffer appropriate for amino acid analyzer. Thehydrolysate can not be stored for greater than one week before usinganalyzed.

EXAMPLES

The Examples which follow are intended as an illustration of certainpreferred embodiments of the invention, and no limitation of theinvention is implied. The isolated cocoa procyanidin oligomers used inthis Example 17, 18, and 19 were isolated using the procedure describedin U.S. Pat. No. 5,554,645 (issued Sep. 10, 1996 to L. Romanczyk et al.)and further purified using the procedure of Method B.

Example 1 Method of Obtaining Cocoa Polyphenol; Cocoa Solids from CocoaBeans

Commercially available cocoa beans having an initial moisture content offrom about 7 to 8 percent by weight were pre-cleaned using an 11″×56″Scalperator (manufactured by Carter Day International, Minneapolis,Minn., USA). Approximately 600 bags of cocoa beans (39,000 kg) werepre-cleaned over a 6.5 hour time period. The beans were fed into theinlet hopper where the flow rate was regulated by a positive feed roll.The beans were fed onto the outside of a rotating wire mesh scalpingreel. The beans passed through the wire mesh reel and subsequentlythrough an air aspiration chamber where light dirt, dust and stringswere aspirated out of the product stream. The beans that did not passthrough the scalping reel were conveyed to the reject stream. Thisreject stream consisted of large clumps of beans, sticks, stones, etc.The amount of resultant reject was approximately 150 kg, or 0.38% of thestarting material. The resulting pre-cleaned product weighed about38,850 kg and was passed to the bean cleaning step.

The pre-cleaned bean products from the Scalperator were then furthercleaned using a Camas International SV4-5 Air Fluidized Bed DensitySeparator (AFBDS, manufactured by Camas International, Pocotello, Id.,USA). About 38,850 kg of cocoa bean products were fed into the AFBDSover a time period of about 6.5 hours. The apparatus removedsubstantially all heavy impurities such as stones, metal, glass, etc.from the beans, as well as lighter unusable materials such as moldy andinfested cocoa beans, resulting in a cleaned bean product whichcontained substantially only usable cocoa beans. The resulting heavyimpurities removed weighed about 50 kg and the light unusable materialsweighed about 151 kg. A total of about 38,649 kg of cleaned beans wasobtained after both the pre-cleaning and cleaning steps described hereinabove (99.1% yield after cleaning).

The cleaned cocoa beans were then passed through a infra-red heatingapparatus. The apparatus used was the Micro Red 20 electric infra-redvibratory Micronizer (manufactured by Micronizing Company (U.K.)Limited, U.K.). The Micronizer was run at a rate of about 1,701kilograms per hour. The depth of beans in the vibrating bed of theMicronizer was about 2 inches or about 2-3 beans deep. The surfacetemperature of the Micronizer was set at about 165° C., resulting in anIBT of about 135° C., for a time ranging from 1 to 1.5 minutes. Thistreatment caused the shells to dry rapidly and separate from the cocoanib. Since substantially all of the cocoa beans fed into the Micronizerwere whole beans and were substantially free of small broken pieces ofbean or shell, no sparks or fires were observed during the infra-redheating step. The broken pieces separated by the vibrating screen priorto the Micronizer were re-introduced into the product stream prior tothe winnowing step.

The beans from the Micronizer had a moisture content of about 3.9% byweight. The beans emerged from the Micronizer at an IBT of about 135° C.and were immediately cooled to a temperature of about 90° C. in aboutthree minutes to minimize additional moisture loss. The total beansavailable after the heating step was about 36,137 kg.

The beans were then subjected to winnowing using a Jupiter Mitra Seitawinnower (manufactured by Jupiter Mitra Seita, Jakarta, Indonesia). Thewinnowing step cracked the beans to loosen the shells and separated thelighter shells from the nibs while at the same time minimizing theamount of nib lost with the shell reject stream. The feed rate into thewinnower was about 1,591 kg per hour. The resultant products includedabout 31,861 kg of usable nibs and 4,276 kg of reject shells. Theoverall yield of usable nibs from starting material was about 81.7%.

The resulting cocoa nibs were pressed using a Dupps 10-6 Pressor(manufactured by The Dupps Company, Germantown, Ohio, USA). A steady,consistent feed of about 1,402 kg per hour of nibs was fed into twoscrew presses to extract butter. The press produced about 16,198 kg ofcocoa butter which contained about 10% cocoa solids, and about 15,663 kgof cocoa solids which contained about 10% butter.

The cocoa butter was further processed using a Sharples P3000 decantingcentrifuge (manufactured by Jenkins Centrifuge Rebuilders, N. KansasCity, Mo., USA). Centrifugation reduced the solids content in the butterto about 1-2% solids and provided about 13,606 kg of butter and 2,592 kgof cocoa solids containing about 40 to 45% butter. The butter containing1-2% solids was further processed using a plate and frame filter(manufactured by Jupiter Mitra Seita) which removed the remaining solidsfrom the butter and provided about 13,271 kg of clear cocoa butter andabout 335 kg of cocoa solids containing 40-45% butter.

The cocoa solids removed from the centrifuge and the filter presscontained about 40-45% fat and were pressed in a batch hydraulic pressto produce 10% fat cocoa cake. This material produced about 1,186 kg ofclear butter and 1,742 kg of cocoa solids.

The total clear butter yield from the incoming beans was 14,456 kg, or37.1%. The total cocoa solids produced from the incoming beans was17,405 kg, or 44.6%.

A sample of the partially defatted cocoa solids cocoa powder, producedaccording to the above-described process from unfermented cocoa beans(fermentation factor 100), contained the following procyanidinconcentrations: total procyanidin 32,743 μg/g, procyanidin 9,433 μg/g,procyanidin dimer 5,929 μg/g, procyanidin trimer 5,356 μg/g, procyanidintramer 4,027 μg/g, procyanidin pentamer 3,168 μg/g, procyanidin hexamer2,131 μg/g, procyanidin heptamer 1,304 μg/g, procyanidin octamer 739μg/g, procyanidin nonamer 439 μg/g.

Example 2 Production of Chocolate Liquor Containing Cocoa Polyphenols

Fair average quality (FAQ) Sulawesi cocoa beans having an initialmoisture content 7.4% by weight and a fermentation factor level of 233(31% slaty, 29% purple, 22% purple brown and 17% brown) were selected asthe starting material. The cocoa beans were then passed through aninfra-red heating apparatus. The apparatus used was an infra-redvibrating micronizer (manufactured by Micronizer Company (U.K.) Limited,U.K.). The feed rate of beans through the infra-red heater and theinfra-red heater bed angle were varied to control the amount of heattreatment the beans received. The amount of time the beans spent in theinfra-red heater (residence time) was determined by the bed angle andthe feed rate. The times used to prepare the materials are listed in theTable 1 below. At the outlet of the micronizer the internal beantemperature (IBT) of the beans was measured, these values are also shownin Table 1.

A 1 kg sample of infra-red heated beans, collected off the infra-redheater at different IBTs, were cracked into smaller pieces. This wasdone to facilitate the separation of the nib from the shell. Thelaboratory piece of equipment used to remove the shell was theLimiprimita Cocoa Breaker made by the John Gordon Co. LTD. of England.The cracked beans were next passed through a laboratory scale winnowingsystem, using a Catador CC-1 manufactured by the John Gordon Co. LTD,England.

The cocoa nibs were next milled into a coarse liquor using a Melangemade by Pascall Engineering Co. LTD, England. This device crushes andgrinds the nibs into a chocolate liquor. The normal operatingtemperature for the liquor in the Melange is approximately 50° C. Thissame process of making nibs to a coarse liquor could be done on a largerproduction scale using other types of mills, such as a Carle & MontanariMill. The cocoa nibs were ground in the Melange for one hour. Theconcentration of cocoa procyanidins was measured for the samplesrelative to the infra-red heated temperatures. These values are given inthe Table 1 below.

TABLE 1 Total Residence Moisture in Procyanidin Procyanidins Time inFinished in Defatted in Defatted Micronizer, Liquor Liquor Liquor IBT °C. (seconds) (%) (μg/g) (μg/g) 107 42 3.9 3,098 39,690 126 82 1.87 1,48728,815 148 156 1.15 695 23,937

Example 3 Chocolate Food Product

A 10 lb. Sigma blade mixer (manufactured by Teledyne Read Co., York,Pa.) was used to mix together ingredients within the concentrationranges set forth below. The selection of the appropriate ingredients andamounts within the given range to prepare a chocolate is readilyperformed by one skilled in the art, without undue experimentation.

% Concentration Ingredient (by weight) Sucrose 40% Chocolate Liquor  7%CP Liquor (Ex. 2) 49% Fat 3.5%  Lecithin 0.5% 

The lecithin and fat were combined and mixed, using a 10 lb. Sigma blademixer until homogeneous. The resulting fat/lecithin mixture was added tothe granulated sucrose in a second 10 lb. Sigma mixer. The sucrose, fatand lecithin were mixed at about 35° C. to about 90° C. untilhomogeneous. The remaining ingredients, including the chocolate liquorof Example 2 having a high concentration of cocoa procyanidins, wereadded and mixed until homogeneous. The resulting mixture was refined toa micrometer particle size of about 20 microns, conched, standardized.The cocoa procyanidin pentameter concentration of the resultingchocolate ranged from about 385 to 472 μg per gram of chocolate.

Peanuts in an amount of approximately 5-30 percent by weight of thefinal product are added to form a chocolate-containing peanut producthigh in cocoa procyanidins and L-arginine.

Example 4 Peanut Butter Food Product

Preroasted peanuts are ground with the addition of salt and sugar asdesired to form peanut butter. While mixing, the cocoa powder of Example1 which has a high cocoa procyanidin content, is added to the mixture inan amount of approximately 2 to 3 percent by weight of the totalmixture. The product is a peanut butter containing cocoa polyphenols andL-arginine.

Example 5 Pharmaceutical Composition

A tablet mixture is prepared which comprises the following ingredients(percentages expressed as weight percent):

Cocoa Powder of Example 1 24.0% L-arginine 5.0% Natural Vanilla Extract1.5% Magnesium Stearate (lubricant) 0.5% Dipac tabletting Sugar 32.0%Xylitol 37.0%

The cocoa powder, vanilla extract and L-arginine are blended together ina food processor for several minutes. The sugars and magnesium stearateare gently mixed together, followed by blending with the cocoapowder/vanilla extract/L-arginine mixture. This material is run througha Manesty Tablet Press (B3B) at maximum pressure and compaction toproduce round tablets (15 mm×5 mm) weighing 1.5 to 1.8 grams.

Example 6 Dark Chocolate

A dark chocolate is prepared in a manner substantially similar to theprocess described in Example 3, using the following general recipe:

-   -   Ingredient Range (wt. %)    -   15-35% Sucrose    -   40-75% CP Liquor of Ex. 2    -   1-10% CP Cocoa Powder of Ex. 1    -   0.01-0.05% Vanillin    -   0.1-1.0% Lecithin

Peanuts in an amount of approximately 5 to 30 percent by weight of thetotal product are added to the dark chocolate.

Example 7 Milk Chocolate

A milk chocolate is prepared in a manner substantially similar to theprocess described in Example 3, using the following general recipe:

-   -   Ingredient Range (wt. %)    -   35-55% Sucrose    -   12-25% Milk Ingredient    -   10-20% CP Liquor of (Ex. 2)    -   15-25% Fat    -   0.1-1.0% Emulsifier

Almonds in an amount of approximately 5 to 30 percent by weight of thetotal product are added to the chocolate.

Example 8 Peanut Butter-Soy Cookie Bar

Enrobed with High CP Dark Chocolate

Ingredient % Range Dark Chocolate 3.4 mg per g chocolate 35 30-40Peanuts 32 30-40 Soy Flour, low fat 11 10-15 Vegetable Oil 5  2-10 Sugar15 10-20 Water 1.5 Salt <1 Caramel Syrup Solution <1 Sodium Bicarbonate<1 Propyl Gallate <1

The peanut butter is prepared by combining the peanuts, sugar, vegetableoil, salt and propyl gallate. Cookie is prepared by combining soy flour,water, vegetable oil and sodium bicarbonate, and baking. The peanutbutter is then extruded onto the baked cookies and then entire bar isenrobed in the high CP dark chocolate.

Based on the cocoa procyanidin, nut procyanidins, and arginine contentof the recipe ingredients, the theoretical procyandin and arginineconcentrations are shown below:

Total Procyanidins 120 mg/100 g Arginine  1.4 g/100 g

Example 9 Dry Drink Mix Containing High CP Cocoa and L-arginine

A dry drink mix containing the cocoa powder from Example 1 havingenhanced levels of cocoa polyphenols (CPs) and L-arginine is madeaccording to the following formulations:

Ingredient % Sugar 59 Skim Milk Powder 20 Malt Powder 1.9 CP CocoaPowder 25-50 mg/g 8.0 cocoa powder Peanut Flour 10.0 Vanillin <0.01Lecithin <0.995 Salt <0.1 Flavoring <0.1

The dry ingredients are batched according to the above formulation andmixed for one hour in a Kitchen Aid Professional Mixer (Model KSM50P)using a wire whip at #2 speed. The lecithin is agglomerated prior to usein the recipe in a Niro-Aeromatic Agglomerator (Model STREA/1).

Based on the cocoa procyanidin, nut procyanidin, and arginine content ofthe recipe ingredients, the theoretical procyanidin and L-arginineconcentrations are shown below:

Procyanidins 200-400 mg/100 g L-arginine      0.9 g/100 g

Example 10 Nut and Seed Bar with Cocoa Extract

Almonds) 30 Pumpkins Seeds 12 Sunflower seeds 5 Sesame seeds 5 Salt <1Butter 10 Corn syrup 7.6 Lecithin <1 Sugar 26 Cocoa extract 4Almonds are lightly toasted in salted butter. Pumpkin seeds, sunflowerseeds, and sesame seeds are added. Butter, corn syrup, lecithin and saltare combined and heated in microwave oven on % power for 1 minute. Sugaris placed in a stainless steel saucepan and cooked on an inductioncooker at full power. When the sugar is almost completely melted, heatis reduced to medium power (290° F.) and the sugar cooked until thesugar is fully melted and honey in color. When the sugar is fullymelted, it is slowly added to the corn syrup/butter mixture and mixed.The nut mix is placed in a stand mix. The syrup is carefully poured intothe nut mix with the paddle on slow speed. The nut/syrup mix is formedinto bars and cooled.

Based on the cocoa procyanidin, nut procyanidin, and arginine content ofthe recipe ingredients, the theoretical procyanidin and arginineconcentrations are shown below:

Procyanidins 1586 mg/100 g Arginine   1.6 g/100 g

Example 11 Peanut, Caramel, Nougat, Bar Enrobed in High CP DarkChocolate

% Formula CP dark chocolate 31.5 Peanuts w/skins 30.0 Caramel 27.0Nougat 11.5

A nougat mixture is prepared that contains 45% peanuts and is slabbedonto a cooling table and cut into rectangular bars. A caramel mixture isprepared that contains about 38% peanuts, cooled, and cut into similarpieces. The nougat is topped with the caramel slab and the whole bar isenrobed in chocolate which contains about 10% peanuts.

Based on the cocoa procyanidin, nut procyanidin, and arginine content ofthe recipe ingredients, the theoretical procyanidin and L-arginineconcentration are shown below:

Procyanidins 260 mg/100 gArginine 1 g/100 g

Example 12 Cocoa Source and Method of Preparation

Several Theobroma cacao genotypes which represent the three recognizedhorticultural races of cocoa (Enriquez, 1967; Engels, 1981) wereobtained from the three major cocoa-producing regions of the world. Alist of those genotypes is shown in Table 2. Other species of Theobromacacao and its closely related genus Herranina will also be suitable foruse herein.

TABLE 2 Description of Theobroma cacao Source Material GENOTYPE ORIGINHORTICULTURAL RACE UIT-1 Malaysia Trinitario Unknown West AfricaForaster ICS-100 Brazil Trinitario (Nicaraguan Criollo ancestor) ICS-39Brazil Trinitario (Nicaraguan Criollo ancestor) UF-613 Brazil TrinitarioEEG-48 Brazil Forastero UF-12 Brazil Trinitario NA-33 Brazil Forastero

Harvested cocoa pods were opened and the underfermented beans with thepulp were removed and freeze-dried. The pulp was manually removed. Thebeans were manually dehulled, and ground to a fine powdery mass with aTEKMAR Mill. The resultant mass was then defatted overnight by Soxhletextraction using redistilled hexane as the solvent. Residual solvent wasremoved from the defatted mass by vacuum at ambient temperature.

Example 12 Procyanidin Extraction Procedures

A. Method 1

Procyanidins were extracted from the defatted, unfermented, freeze-driedcocoa beans of Example 11 using a modification of the method describedby Jalal and Collin (1977). Procyanidins were extracted from 50 grambatches of the defatted cocoa mass with 2 400 mL 70% acetone/deionizedwater followed by 400 mL 70% methanol/deionized water. The extracts werepooled and the solvents were removed by evaporation at 45° C. with arotary evaporator held under partial vacuum. The resultant aqueous phasewas diluted to 1 L with deionized water and extracted 2 times with 400mL CHCl₃. The solvent phase was discarded. The aqueous phase was thenextracted 4 times with 500 mL ethyl acetate. Any resultant emulsionswere broken by centrifugation on a Sorvall RC 28S centrifuge operated at2,000× gravity for 30 min. at 10° C. with a rotary evaporator held underpartial vacuum. The resultant aqueous phase was frozen in liquid N₂,followed by freeze drying on a LABCONCO Freeze Dry System. The yields ofcrude procyanidins that were obtained from the different cocoa genotypesare listed Table 3.

TABLE 3 Crude Procyanidin Yields GENOTYPE ORIGIN YIELDS (g) UIT-1Malaysia 3.81 Unknown West Africa 2.55 ICS-100 Brazil 3.42 ICS-39 Brazil3.45 UF-613 Brazil 2.98 EEG-48 Brazil 3.15 UF-12 Brazil 1.21 NA-33Brazil 2.23

B. Method 2

Alternatively, procyanidins are extracted from the defatted,unfermented, freeze-dried cocoa beans of Example 1 with 70% aqueousacetone. Ten grams of defatted material was slurried with 100 mL solventfor 5-10 min. The slurry was centrifuged for 15 min. at 4° C. at 3000×gravity and the supernatant was passed through glass wool. The filtratewas subjected to distillation under partial vacuum and the resultantaqueous phase was frozen in liquid N₂, followed by freeze drying on aLABCONCO Freeze Dry System. The yields of crude procyanidins ranged from15-20%.

It is believed that the differences in crude yields reflected variationsencountered with different genotypes, geographical origin, horticulturalrace, and method of preparation.

Example 13 Partial Purification of Cocoa Procyanidins

A. Gel Permeation Chromatography

The procyanidins obtained from Example 12 were partially purified byliquid chromatography on Sephadex LH-20 (28×2.5 cm). Separations wereaided by a step gradient from deionized water into methanol. The initialgradient composition started with 15% methanol in deionized water whichwas followed step wise every 30 min. with 25% methanol in deionizedwater, 35% methanol in deionized water, 70% methanol in deionized water,and finally 100% methanol. The effluent following the elution of thexanthine alkaloids (caffeine and theobromine) was collected as a singlefraction. The fraction yielded a xanthine alkaloid-free subfractionwhich was submitted to further subfractionation to yield fivesubfractions designated MM2A through MM2E. The solvent was removed fromeach subfraction by evaporation at 45° C. with a rotary evaporator heldunder partial vacuum. The resultant aqueous phase was frozen in liquidN₂ and freeze dried overnight on a LABCONCO Freeze Dry System. Arepresentative gel permeation chromatogram showing the fractionation wasobtained. Approximately, 100 mg of material was subfractionated in thismanner.

Chromatographic Conditions: Column; 28×2.5 cm Sephadex LH-20,

Mobile Phase Methanol/Water Step Gradient, 15:85, 25:75, 35:65, 70:30,100:0 Stepped at ½ Hour Intervals, Flow Rate; 1.5 mL/min,Detector; UV at λ₁=254 nm and λ₂=365 nm, Chart Speed: 9.5 mm/min, ColumnLoad; 120 mg.

B. Semi-preparative High Performance Liquid Chromatography (HPLC)

Method 1. Reverse Phase Separation

Procyanidins obtained from Example 2 and/or 3A were partially purifiedby semi-preparative HPLC. A Hewlett Packard 1050 HPLC System equippedwith a variable wavelength detector, Rheodyne 7010 injection valve with1 mL injection loop was assembled with a Pharmacia FRAC-100 FractionCollector. Separations were effected on a Phenomenex Ultracarb™ 10μ ODScolumn (250×22.5 mm) connected with a Phenomenex 10μ ODS Ultracarb™(60×10 mm) guard column. The mobile phase composition was A=water;B=methanol used under the following linear gradient conditions; [Time, %A]; (0, 85), (60, 50), (90, 0), and (110, 0) at a flow rate of 5 mL/min.Compounds were detected by UV at 254 nm.

A representative semi-preparative HPLC trace was obtained for theseparation of procyanidins present in fraction D+E. Individual peaks orselect chromatographic regions were collected on timed intervals ormanually by fraction collection for further purification and subsequentevaluation. Injection loads ranged from 25-100 mg of material.

Method 2. Normal Phase Separation

Procyanidin extracts obtained from Examples 2 and/or 3A were partiallypurified by semi-preparative HPLC. A Hewlett Packard 1050 HPLC system,Millipore-Waters Model 480 LC detector set at 254 nm was assembled witha Pharmacia Frac-100 Fraction Collector set in peak mode. Separationswere effected on a Supelco 5 μm Supelcosil LC-Si column (250×10 mm)connected with a Supelco 5 μm Supelguard LC-Si guard column (20×4.6 mm).Procyanidins were eluted by a linear gradient under the followingconditions: (Time, % A, % B); (0, 82, 14), (30, 67.6, 28.4), (60, 46,50, (65, 46, 50), (65, 10, 86), (70, 10, 86) followed by a 10 min.re-equilibration. Mobile phase composition was A=dichloromethane;B=methanol; and C=acetic acid:water (1:1). A flow rate of 3 mL/min wasused. Components were detected by UV at 254 nm, and recorded on a Kipp &Zonan BD41 recorder. Injection volumes ranged from 100-250 μL of 10 mgof procyanidin extracts dissolved in 0.25 mL 70% aqueous acetone. Arepresentative semi-preparative HPLC trace was obtained. Individualpeaks or select chromatographic regions were collected on timedintervals or manually by fraction collection for further purificationand subsequent evaluation.

HPLC Conditions:250×10 mm Supelco Supelcosil LC-si

-   -   (5 μm) Semipreparative Column    -   20×4.6 mm Supelco Supelcosil LC-Si    -   (5 μm) Guard Column    -   Detector: Waters LC    -   Spectrophotometer Model 480 @ 254 nm    -   Flow rate: 3 mL/min,    -   Column Temperature: ambient,    -   Injection: 250 μL of 70% aqueous acetone extract

Gradient: Acetic Acid: Time (min) C₂Cl₂ Methanol H₂O (1:1) 0 82 14 4 3067.6 28.4 4 60 46 50 4 65 10 86 4 70 10 86 4

Example 14 HPLC Purification Methods

Method A. GPC Purification

Procyanidins obtained as in Example 12 were partially purified by liquidchromatography on Sephadex LH 20 (72.5×2.5 cm), using 100% methanol asthe eluting solvent, at a flow rate of 3.5 mL/min. Fractions of theeluent were collected after the first 1.5 hours, and the fractions wereconcentrated by a rotary evaporator, redissolved in water, andfreeze-dried. These fractions were referred to as pentamer-enrichedfractions. Approximately 2.00 g of the extract obtained from Example 2was subfractionated in this manner. The results are shown in Table 4.

TABLE 4 Composition of Fractions Obtained: Fraction Monomer Dimer TrimerTetramer Pentamer Hexamer (Time) (% Area) (% Area) (% Area) (% Area) (%Area) (% Area) 1:15 73 8 16 3 ND ND 1:44 67 19 10 3 1 tr 2:13 30 29 2411 4 1 2:42 2 16 31 28 15 6 3:11 1 12 17 25 22 13 3:40 tr 18 13 18 20 154:09 tr 6 8 17 21 19 1:15 ND ND ND ND ND ND 1:44 tr tr tr tr tr tr 2:131 tr tr tr tr tr 2:42 2 tr tr tr tr tr 3:11 7 1 tr tr tr tr 3:40 10 2 2tr tr tr 4:09 14 4 4 2 tr tr ND = not detected tr = trace amount

Method B. Normal Phase Separation

Procyanidins obtained as Example 12 were separated, purified by normalphase chromatography on Supelcosil LC-Si, 100 Á, 5 μm (250×4.6 mm), at aflow rate of 1.0 mL/min, or, in the alternative, Lichrosphere∘ Silica100, 100 Á, 5 μm (235×3.2 mm), at a flow rate of 0.5 mL/min. Separationswere aided by a step gradient under the following conditions: (Time, %A, % B); (0, 82, 14), (30, 67.6 28.4), (60, 46, 50), (65, 10, 86), (70,10, 86). Mobile phase composition was A=dichloromethane; B=methanol; andC=acetic acidwater (1:1). Components were detected by fluorescence whereλ_(ex)=276 nm and λ_(em)=316 nm, and by UV at 280 nm. The injectionvolume was 5.0 μL (20 mg/mL) of the procyanidins obtained from Example2.

In the alternative, separations were aided by a step gradient under thefollowing conditions: (Time, % A, % B); (0, 76, 20); (25, 46, 50); (30,10, 86). Mobile phase composition was A=dichloromethane; B=methanol; andC=acetic acidwater (1:1).

Method C. Reverse-Phase Separation

Procyanidins obtained as in Example 12 were separated and purified byreverse phase chromatography on Hewlett Packard Hypersil ODS 5 μm(200×2.1 mm), and a Hewlett Packard Hypersil ODS 5 μm guard column(20×2.1 mm). The procyanidins were eluted with a linear gradient of 20%B into A in 20 minutes, followed by a column wash with 100% B at a flowrate of 9.3 mL/min. The mobile phase composition was a degassed mixtureof B=1.0% acetic acid in methanol and A=2.0% acetic acid in nanopurewater. Components were detected by UV at 280 nm, and fluorescence whereζ_(ex)=276 nm and λ_(em)=316 nm; the injection volume was 2.0 μL (20mg/mL).

Example 15 HPLC Separation of Pentamer Enriched Fractions

Method A. Semi-Preparative Normal Phase HPLC

The pentamer-enriched fractions were further purified bysemi-preparative normal phase HPLC by a Hewlett Packard 1050 HPLC systemequipped with a Millipore-Waters model 480 LC detector set at 254 nmwhich was assembled with a Pharmacia Frac-100 Fraction Collector set topeak mode. Separations were effected on a Supelco 5 μm Supelcosel LC-Si,100 Å column (250×10 mm) connected with a Supelco 5μ Supelguard LC-Siguard column (20×4.6 mm). Procyanidins were eluted by a linear gradientunder the following conditions: (Time % A, % B); (0, 82, 14), (30, 67.6,28.4), (60, 46, 50), (65, 10, 86), (70, 10, 86) followed by a 10 minutere-equilibration. Mobile phase composition was A=dichloromethane;B=methanol; and C=acetic acid:water (1:1). A flow rate of 3 mL/min wasused. Components were detected by UV at 254 nm and recorded on a Kipp &Zonan BD41 recorder. Injection volumes ranged from 100-250 μl of 10 mgof procyanidin extracts dissolved in 0.25 mL of 70% aqueous acetone.Individual peaks or select chromatographic regions were collected ontimed intervals or manually by fraction collection for furtherpurification and subsequent evaluation.

HPLC conditions:250×100 mm Supelco Supelcosil LC-Si

-   -   (5 μm) Semipreparative Column    -   20×4.6 mm Supelco Supelcosil LC-Si    -   (5 μm) Guard Column    -   Detector: Waters LC    -   Spectrophotometer Model 480 @ 254 nm    -   Flow Rates: 3 mL/min.    -   Column Temperature: ambient    -   Injection: 250 μL of pentamer enriched extract

acetic acid: Gradient: CH₂Cl₂ methanol water (1:1) 0 62 14 4 30 67.628.4 4 60 46 50 4 65 10 86 4 70 10 86 4

Method B. Reverse Phase Separation

Procyanidin extracts obtained as in Example 15 were filtered through a0.45μ nylon filter and analyzed by a Hewlett Packard 1090 ternary phaseHPLC system equipped with a Diode Array detector and a HP model 1046AProgrammable Fluorescence Detector. Separations were effected at 45° C.on a Hewlett Packard 5μ Hypersil ODS column (200×2.1 mm). Theprocyanidins were eluted with a linear gradient of 60% B into A followedby a column wash with B at a flow rate of 0.3 mL/min. The mobile phasecomposition was a de-gassed mixture of B=0.5% acetic acid in methanoland A=0.5% acetic acid in nanopure water. Acetic acid levels in A and Bmobile phases can be increased to 2%. Components were detected byfluorescence, where λ_(ex)=276 nm and λ_(em)=316 nm, and by UV at 280nm. Concentrations of (+)-catechin and (−)-epicatechin were determinedrelative to reference standard solutions. Procyanidin levels wereestimated by using the response factor for (−)-epicatechin.

Method C. Normal Phase Separation

Pentamer-enriched procyanidin extracts obtained as in Example 15 werefiltered through a 9.45μ nylon filter and analyzed by a Hewlett Packard1090 Series II HPLC system equipped with a HP Model 1046A ProgrammableFluorescence detector and Diode Array detector. Separations wereeffected at 37° C. on a 5μ Phenomenex Lichrosphere∘ Silica 100 column(250×3.2 mm) connected to a Supelco Supelguard LC-Si 5μ guard column(20×4.6 mm). Procyanidins were eluted by linear gradient under thefollowing conditions: (time, % A, % B); (0, 82, 14), (30, 67.6, 28.4),(60, 46, 50), (65, 10, 86), (7, 10, 86), followed by an 8 minutere-equilibration. Mobile phase composition was A=dichloromethane,B=methanol, and C=acetic acid:water at a volume ratio of 1:1. A flowrate of 0.5 mL/min was used. Components were detected by fluorescence,where λ_(ex)=276 nm and λ_(em)=316 nm or by UV at 280 nm. Arepresentative HPLC chromatogram showing the separation of the variousprocyanidins was obtained for one genotype. Similar HPLC profiles wereobtained from other Theobroma, Herrania and/or their inter- orintra-specific crosses.

HPLC conditions:

250×3.2 mm henomenex Lichrosphere∘ Silica 100

column (5μ) 20×4.6 mm Supelco Supelguard LC-Si (5μ)

guard column

Detectors: Photodiode Array @ 280 nm

Fluorescence λ_(ex)=276 nm and λ_(em)=316 nm

Flow rate: 9/5 mL/min.

Column temperature: 37° C.

acetic acid:

Gradient: CH₂Cl₂ methanol water (1:1) 0 62 14 4 30 67.6 28.4 4 60 46 504 65 10 86 4 70 10 86 4

Method D. Preparative Normal Phase Separation

The pentamer-enriched fractions obtained as in Example 5 were furtherpurified by preparative normal phase chromatography by modifying themethod of Riguad et al., (J. Chrom. 654:255-260. (1993)

Separations were affected at ambient temperature on a 5μ SupelcosilLC-Si 100 Å column (50×2 cm) with an appropriate guard column.Procyanidins were eluted by a linear gradient under the followingconditions: (time, % A, % B, flow rate); (0, 92.5, 7.5, 10); (10, 92.5,7.5, 40); (30, 91.5, 18.5, 40); (145, 88, 22, 40); (150, 24, 86, 40);(155, 24, 86, 50); (180, 0, 100, 50). Prior to use, the mobile phasecomponents were mixed by the following protocol:

Solvent A preparation (82% CH₂Cl₂, 14% methanol, 2% acetic acid, 2%water):1. Measure 80 mL of water and dispense into a 4 L bottle.2. Measure 80 mL of water and dispense into the same 4 L bottle.3. Measure 560 mL of methanol and dispense into the same 4 L bottle.4. Measure 3280 mL of methylene chloride and dispense into the same 4 Lbottle.5. Cap the bottle and mix well.6. Purge the mixture with high purity helium for 5-10 minutes to degas.Repeat steps 1-6 two times to yield 8 volumes of solvent A. Solvent Bpreparation (96% methanol, 2% acetic acid, 2% water):1. Measure 80 mL of water and dispense into a 4 L bottle.2. Measure 80 mL of acetic acid and dispense into the same 4 L bottle.3. Measure 3840 mL of methanol and dispense into the same 4 L bottle.4. Cap the bottle and mix well.5. Purge the mixture with high purity helium for 5-10 minutes to degas.Repeat steps 1-5 to yield 4 volumes of solvent B. Mobile phasecomposition was A=methylene chloride with 2% acetic acid and 2% water;B=methanol with 2% acetic acid and 2% water. The column load was 0.7 gin 7 mL. Components were detected by UV at 254 nm. A typical preparativenormal phase HPLC separation of cocoa procyanidins was obtained.

HPLC Conditions:

Column: 50×2 cm 5μ Supercosil LC-Si run @ ambient temperature.Mobile Phase: A=Methylene Chloride with 2% Acetic Acid and 2% Water.

B=Methanol with 2% Acetic Acid and 2% Water.

Gradient/Flow Profile

TIME FLOW RATE (MIN) % A % B (mL/min) 0 92.5 7.5 10 10 92.5 7.5 40 3091.5 8.5 40 145 88.0 22.0 40 150 24.0 86.0 40 155 24.0 86.0 50 180 0100.0 50

Example 16 Purification of Oligomeric Fractions

Method A. Purification by Semi-Preparative Reverse Phase HPLC

Procyanidins obtained from Example 15, Methods A and B and D, werefurther separated to obtain experimental quantities of the oligomers. AHewlett Packard 1050 HPLC system equipped with a variable wavelengthdetector, Rheodyne 7010 injection valve with 1 mL injection loop wasassembled with a pharmacia FRAC-100 Fraction Collector. Separations wereeffected on a Phenomenex Ultracarb° 10μ ODS column (250×22.5 mm)connected with a Phenomenex 10μ ODS Ultracarb® (60×10 mm) guard column.The mobile phase composition was A=water; B=methanol used under thefollowing linear gradient conditions: (time, % A); (0, 85), (60, 50),(900) and (110, 0) at a flow rate of 5 mL/min. Individual peaks orselect chromatographic regions were collected on timed intervals ormanually by fraction collection for further evaluation by MADLY-OF/MSand NKr. Injection loads ranged from 25-100 mg of material. Arepresentative elution profile was obtained.

Method B. Modified Semi-Preparative HPLC

Procyanidins obtained from Example 15, Methods A and B and D werefurther separated to obtain experimental quantities of like oligomers orfurther structural identification and elucidation (e.g., Example 15, 18,19, and 20). Supelcosil LC-Si 5μ column (250×10 mm) with a SupelcosilLC-Si 5μ (20×2 mm) guard column. The separations were effected at a flowrate of 3.0 mL/min, at ambient temperature. The mobile phase compositionwas A=dichloromethane; B=methanol; and C=acetic acid:water (1:1); usedunder the following linear gradient conditions: (time, % A, % B); (0,82, 14), (22, 74, 21), (60, 74, 21); (60, 74, 50, 4); (61, 82, 14),followed by column re-equilibration for 7 minutes. Injection volumeswere 60 μL containing 12 mg of enriched pentamer. Components weredetected by UV at 280 nm. A representative elution profile was obtained.

Example 17 Assessment of Nitric Oxide Synthase Activity

The culture medium used was Medium 200 (Cascade Biologics Inc.)supplemented with Low Serum Growth Supplement (Cascade Biologics Inc.)and 20% fetal calf serum (DAP).

The cocoa procyanidins evaluated were the epicatechin monomer, thedimer, the trimer, the tetramer, the pentamer, and the heptamer. Thenitrite content of cocoa procyanidins were evaluated. At the maximalconcentration used (100

g/ml), no nitrite was detected.

Human umbilical vein endothelial cells (HUVEC) were purchased at primaryculture stage from Cascade Biologics Inc. (Portland). Cells werecultured in Medium supplemented with Low Serum Growth Supplement (LSGS)and 20% Fetal Calf Serum (FCS) in 75 cm² flasks. The cells were seededfor a week following treatment with trypsin-EDTA (2 ml/flask) at 37° C.under 5% CO₂ atmosphere. Trypsin was neutralized upon addition of 3 ml.FCS.

The cell suspension was centrifuged for 10 min. at 1200 rpm and the cellpellet was resuspended in the culture medium described above.

Nitric oxide synthase activity was assessed by measuring the nitriteconcentration in the culture medium. HUVEC were used between passage 2to 13. Cells were cultured in 24-well culture plates at theconcentration of 5×10⁵ cells ml in Medium 200 containing LSGS (300 μlper well), and 20% CS. After a 24 hour to 48 hour incubation period at37° C. under a 5% CO₂ atmosphere, the cells were used confluent (2.5×10⁶cells). Medium was removed and fresh was added.

Cocoa procyanidin monomer and oligomers were added to the culture mediumat 100 μg/ml, 10 μg/ml or 1 μg/ml (final concentration). Controlsconsisted of cells cultured without the procyanidins. Referencecompounds included acetylcholine, ionomycin (NO synthase stimulator viacalcium entry), lipopolysaccharide (NO synthase inductor), and N-methylL-arginine acetate (inhibitor of NO synthase). The reference compoundswere used in order to evidence nitrite production from endothelial NOsynthesis.

NO production was estimated by measurement of nitrite (NO₂)concentration in culture supernatants according to the Griess reaction.Briefly, 50 μl of conditional medium were incubated with 150 μl ofGriess reagent (1% sulfanilamide in 30% acetic acid/0.1%N-(1-naphtyl)-ethylenediamine dihydrochloride in 60% acetic acid) atroom temperature for 2 min. The absorbance at 540 nm was determined in aLabsystems MCC/340 multiskan. Nitrite concentration was determined byusing sodium nitrite as standard and analyzed using ΔSOFT 2.12 software.The cell-free medium and 100 μg/ml of procyanidins contained nodetectable nitrite concentrations.

Raw Data¹: Showing the Effect of Cocoa Procyanidins On Nitric OxideProduction by HUVEC ¹Results are expressed as μmol nitrite/10⁶ cells/48h

(13 Experiments)

Exp. 1 Exp. 2 Exp. 3 Mean 

SD Treatment Control 2.6 2.9 2.4 2.6 0.3 Acetylcholine 10⁻⁵ M 2.5 2.62.8 2.6 0.2 Ionomycin 1 μM 7.8 6.3 5.4 6.5 1.2 Ionomycin 1 μM + LNMA 1.81.9 2 1.9 0.1 1 μM LPS 100 mg/ml 15.6 14.3 13.2 14.4 1.2 LPS 100 mg/ml +LNMA 2.2 2.3 2.6 2.4 0.2 1 μM Monomer  1 μg/ml 2.3 2.5 2.6 2.5 0.2  10μg/ml 2.7 2.8 2.6 2.7 0.1 100 μg/ml 5.4 5.9 5.7 5.7 0.3 Dimer  1 μg/ml2.5 2.7 2.8 2.7 0.2  10 μg/ml 3.8 4.1 3.6 3.8 0.3 100 μg/ml 15.1 16.417.2 16.2 0.1 Trimer  1 μg/ml 3.1 3.3 3.1 3.2 0.1  10 μg/ml 2.9 3.3 2.73.0 0.3 100 μg/ml 11.5 11.8 11.9 11.7 0.2 Tetramer  1 μg/ml 3 3.2 3.33.2 0.2  10 μg/ml 3.6 3.7 4.1 3.8 0.3 100 μg/ml 8.9 9.3 9.2 9.1 0.2Pentamer  1 μg/ml 1.3 1.4 1.3 1.3 0.1  10 μg/ml 2.9 3.4 3 3.1 0.3 100μg/ml 8.8 9.5 9.7 9.3 0.5 Hexamer  1 μg/ml 2.1 2.4 2.4 2.2 0.2  10 μg/ml4.8 5.6 4.3 4.9 0.7 100 μg/ml 9.1 10.4 9.5 9.7 0.7

Unstimulated HUVEC produced 2.6±0.3 μM NO over the 48 hour incubationperiod. Acetylcholine at 10 μM was ineffective in inducing nitric oxideproduction by HUVEC. In contrast, ionomycin (1 MM) and lipolysaccharide(100 ng/ml) evoked a marked production of nitric oxide. This productionof nitric oxide by HUVEC was blocked when N-methyl L-arginine was addedto the incubation medium.

The cocoa procyanidin dimer, pentamer, and heptamer evoked adose-dependent production of NO from HUVEC. Maximum production wasobserved at the highest concentration tested, i.e., 100 μg/ml. Theprocyanidin monomer, trimer, and tetramer also evoked a markedproduction but only at 100 μg/ml concentration. The potency of thevarious procyanidins, considering the highest dose used is as follows:dimer>trimer>heptamer=pentmer=tetramer>monomer.

Example 18 Nitric Oxide-Dependent Hypertension in the Guinea-Pig

Guinea-pigs (around 400 g body weight, male and female) wereanesthetized upon injection of 40 mg/kg sodium pentobarbital. Thecarotid artery was canulated for monitoring of the arterial bloodpressure through a Gould pressure transducer (Model P500). Each of thesix cocoa procyanidins, at concentrations of 1, 3, 10, and 25 mg/kg, wasinjected intravenously through the jugular vein. Alterations in bloodpressure were recorded on a polygraph.

In preliminary experiments (2 animals), it was determined that the doseof 100 mg/kg could not be used since the vehicle containing DMSO had adirect effect on mean arterial blood pressure. No marked effect of thevehicle containing DMSO was noted when the dose of 25 mg/kg of the cocoaprocyanidin was used (15±5%).

The effects of administering of 1, 3, 10, or 25 mg/kg cocoa procyanidinson arterial blood pressure of anesthetized guinea-pigs was investigated.Upon intravenous injection, the procyanidins monomer and dimer evoked adecrease in blood pressure of about 20%, i.e., not markedly differentfrom injection of solvent alone (15±5%, n=5). In contrast, the cocoaprocyanidin trimer, pentamer, and hexamer (10 mg/kg) induced markeddecreases in arterial blood pressure, i.e., up to 62-85% for the cocoaprocyanidin, tetramer and hexamer. The rank of potency of the variouscocoa procyanidins, considering the highest dose used, was as follows:hexamer=tetramer>pentamer>trimer.

TABLE 5 Raw Data: Effect of Cocoa Extracts On The Arterial BloodPressure of Anesthetized Guinea-Pigs (6 experiments) Dose Hypertension(%)* Procyanidin mg/kg Exp 1 Exp 2 Exp 3 Exp 4 Exp 5 Exp 6 Monomer 155.32 70.31 70.27 75.99 71.92 77.97 3 62.99 72.62 74.59 73.33 76.0470.44 10 58.95 66.53 70.06 65.79 70.69 66.21 25 46.93 55.14 63.08 53.9565.91 58.66 Dimer 1 74.47 90.15 92.14 89.74 96.83 94.13 3 77.64 86.2995.29 91.74 100.6 87.08 10 90.76 94.3 99.59 90.59 106.7 97.24 25 88.1495.33 106.6 88.02 110.6 94.02 Trimer 1 75.93 71.61 74.18 74.05 76.4673.23 3 94.99 100 95.05 100.7 92.47 104.8 10 74.34 72.38 73.26 78.94 8070.37 25 12.27 60.42 12.31 11.6 12.23 12.56 Tetramer 1 96.86 89.19 80.3182.47 77.64 109.9 3 101.2 95.14 102.1 99.15 104.9 93.26 10 96.53 0.957.75 12.75 7.53 102.8 25 21.7 3.45 9.61 15.21 9.07 20.83 Pentmer 1 83.4182.57 80.81 81.57 77.55 83.05 3 79.85 80.33 81.47 73.75 74.18 83.43 1087.72 83.56 82.49 75 79.81 89.35 25 59.8 21.13 29.94 21.82 20.27 30.5Hexamer 1 90.24 64.23 93.97 95.97 86.35 94.68 3 74.15 81.18 83.55 80.7369.14 71.97 10 68.8 85.41 69.49 68.04 70.85 71.47 25 25.27 11.2 19.3426.15 25.09 26.6

A comparison of the in vitro and in vivo results shows the followingrank of potency for the cocoa procyanidins:

NO production (100 μg/ml):dimer>trimer>hexamer=pentamer=tetramer>monomer

NO production (10 μg/ml): pentamer, tetramer and dimer (poor induction)

Hypotension: hexamer=tetramer>pentamer>trimer>dimer=monomer.

Except for the dimer, the heptamer or tetramer which induce NOproduction at the lower dose, were the most effective for inducing dropin arterial blood pressure. These results suggest that cocoaprocyanidins can induce in vitro NO production related to an in vivo.

Example 19

Aortic rings from New Zealand white rabbits were set up in 20 ml organbaths. Endothelium dependent relaxation (EDR) to a single dose of (10⁻⁵M) cocoa procyanidin and acetylcholine (Ach) were demonstrated inparallel rings pre-contracted with cocoa procyanidins norepinephine (NE)(10⁻⁵ M). The cocoa procyanidins tested were the monomer, dimer, trimer,tetramer, pentamer, hexamer. Of these, only the pentamer, hexamer, andheptamer demonstrated vasorelaxing activity. Of the two cocoaprocyanidin mixtures tested, only the one containing pentamer to decamer(combo 2) showed significant EDR, whereas the other mixture (combo 1)which contained monomer to tetramer had no effect on vascular tone.

Both combo 2 and the pentamer were then used to demonstrate dosedependent vasorelaxation (10⁻⁸ to 10⁻⁵ M). Rings were incubated for 30′with these cocoa procyanidins (10⁻⁵M) and re-tested with Ach and cocoaprocyanidins (10⁻⁷ to 10⁻⁴). Incubation of the tissue with these cocoaprocyanadin(s) attenuated EDR evoked by both the cocoa procyanidins andAch acutely. The maximum relaxation to Ach pre-incubation, 49±5%; postpentamer incubation, 2±1.4%; post combo 2 incubation, 0.8±0.8%: topentamer pre-incubation, 46.5±4.5%; post incubation, 6.8±3.2%: to combo2 pre-incubation, 54.3±7%; post incubation, 1.7±1.1%, n=5).

The effect of incubation with both cocoa procyanidins (i.e., abolitionof EDR) was restored partially by incubating the tissues with L-arginine(10⁻⁴ M) for 30′ (max relaxation, to Ach: post L-arginine, 21.5±6%; postcombo 2, post L-arginine, 13.7±2.6%: to pentamer; post pentamerincubation, post L-arginine, 18.3±5.8%: to combo 2; post combo 2incubation, post L-arginine, 5.5±2.8%, n=5) The results suggest thatdepletion of substrate for NO synthase may account for this effect.

The above results are shown in Tables 6 to 9

TABLE 6 Preliminary Experiments On The Effect Of Cocoa Procyanidins OnRabbits Aortic Rings Ach Monomer Dimer Trimer Tetramer % relaxation 68 00 0 40 Monomer- Pentamer- Pentamer Hexamer Heptamer Textramer Decamer %relaxation 93 68 47 0 55

TABLE 7 Dose Response Due To Acute Exposure To Cocoa Procyanidins Ach %Relaxation Pentamer % Relaxation Dose (Log mol/1) 10⁻⁷ 10⁻⁶ 10⁻⁵ 10⁻⁷10⁻⁶ 10⁻⁵ % Relaxation 15.2 ± 2.6 42.2 ± 4.2 49 ± 5.1 1.85 ± 0.2 8.3 ±2.5 46.5 ± 4.5 Monomer-Tetramer Pentamer-Decamer % Relaxation %Relaxation Dose (Log mol/1) 10⁻⁷ 10⁻⁶ 10⁻⁵ 10⁻⁷ 10⁻⁶ 10⁻⁵ % RelaxationZero 1.4 ± 0.6 25.5 ± 8.2 54.3 ± 7

TABLE 8 Effect Of Incubation With Cocoa Procyanidins and L-arginine Ach% Relaxation Dose (Log mol/1) 10⁻⁸ 10⁻⁷ 10⁻⁶ 10⁻⁵ Pentamer 0 0  2.1 +1.4 0 Pentamer and 0.53 ± 0.5 4.5 ± 1.0 14.4 ± 5.8 21.5 ± 5.7 L-argininePentamer % Relaxation Dose (Log mol/1) 10⁸ 10⁻⁷ 10⁻⁶ 10⁻⁵ Pentamer 0 0 06.8 ± 3  Pentamer and 0 0.8 ± 0.8 5.6 ± 2.5 18.3 ± 5.7 L-arginine

TABLE 9 Dose (log mol/1) 10⁻⁸ 10⁻⁷ 10⁻⁶ 10⁻⁵ Ach % Relaxation Pentamer-0 0.43 ± 0.4 0.83 ± 0.8 0 Decamer Pentamer- 3.6 ± 0.8  6.4 ± 1.2 11.3 ±2.4 13 .7 ± 2.6 Decamer and L-arginine Pentamer % Relaxation Pentamer- 00 0.58 ± 0.58 1.7 ± 1.1 Decamer Pentamer- 0 1.4 ± 1.4 3.4 ± 1.9 5.5 ±2.8 Decamer and L-arginine

The above findings demonstrate that only cocoa procyanidins above thetrimer are capable of causing vasorelaxation and that cocoa procyanidinshave discrete effects on vascular tone which are unlikely to beassociated with antioxidant activity.

Other variations and modifications, which will be obvious to thoseskilled in the art, are within the scope and teachings of thisinvention. This invention is not to be limited except as set forth inthe following claims.

1-30. (canceled)
 31. A composition comprising (i) at least one cocoapolyphenol and (ii) L-arginine, in a combined amount effective to inducea physiological increase in nitric oxide (NO) production upon oraladministration to a mammal.
 32. The composition of claim 31, wherein thecomposition is edible or ingestible.
 33. The composition of claim 31,wherein the composition is a dietary supplement.
 34. The composition ofclaim 31, wherein the composition is a pharmaceutical composition. 35.The composition of claim 33, wherein the composition is a tablet. 36.The composition of claim 34, wherein the composition is a tablet. 37.The composition of claim 31, wherein the composition is a dry drink mix.38. The composition of claim 33, wherein the composition is a dry drinkmix.
 39. The composition of claim 31, wherein the cocoa polyphenol isprovided within a cocoa ingredient selected from the group consisting ofa shell-free cocoa nib, a chocolate liquor, partially defatted cocoasolids, fully defatted cocoa solids and a cocoa powder.
 40. Thecomposition of claim 31, wherein the cocoa polyphenol is provided withina cocoa extract.
 41. The composition of claim 31, wherein the cocoapolyphenol is a synthesized cocoa polyphenol compound or a mixturethereof and/or a derivative thereof.
 42. The composition of claim 31,wherein the L-arginine is in the form of a peanut-based component. 43.The composition of claim 31, wherein the L-arginine is synthesized. 44.The composition of claim 31, wherein the cocoa polyphenol is present inan amount of about 1 μg to about 10 g per unit dose.
 45. The compositionof claim 31, wherein the L-arginine is present in an amount of 1 μg toabout 10 g per unit dose.
 46. A composition in a unit dosage form fortherapeutic induction of vasorelaxation upon human or veterinary animalconsumption comprising: (i) cocoa polyphenols in an amount of at least 1mg/g, and (ii) L-arginine in the amount of at least 10 mg/g, with theproviso that when the cocoa polyphenols are provided within a cocoaingredient, the amount of L-arginine is greater than that provided withthe cocoa ingredient; wherein the cocoa polyphenols compriseepicatechin, catechin and procyanidin oligomers 2 to 10, the compositioncomprises up to 3 g of the cocoa polyphenols per unit dose, and the unitdose is effective to induce endothelium-dependent vasorelaxation in ahuman or a veterinary animal.
 47. The composition of claim 46, whereinthe cocoa polyphenols are provided within a cocoa extract.
 48. Thecomposition of claim 47, wherein the cocoa polyphenols are present in anamount of at least 3 mg/g.
 49. The composition of claim 46, wherein thecocoa polyphenols are provided within a cocoa ingredient selected fromthe group consisting of a shell-free cocoa nib, a chocolate liquor,partially defatted cocoa solids, fully defatted cocoa solids and a cocoapowder.
 50. The composition of claim 46, wherein the cocoa polyphenolsare present in an amount of at least 1.25 mg/g.
 51. The composition ofclaim 46, wherein the cocoa polyphenols are present in an amount of atleast 1.5 mg/g.
 52. The composition of claim 46, wherein the cocoapolyphenols are present in an amount of at least 2 mg/g.
 53. Thecomposition of claim 46, wherein the cocoa polyphenols are present in anamount of at least 5 mg/g.
 54. The composition of claim 46, wherein thecomposition is a pharmaceutical composition.
 55. The composition ofclaim 46, wherein the composition is a dietary supplement.
 56. Thecomposition of claim 54, wherein the composition is a tablet.
 57. Thecomposition of claim 55, wherein the composition is a tablet.
 58. Thecomposition of claim 46 wherein the composition is a dry drink mix. 59.The composition of claim 55, wherein the composition is a dry drink mix.